Biochimica et Biophysica Acta, 473 (1977) 1-38 © Elsevier/North-Holland Biomedical Press BBA 87034

THE

REVERSE

TRANSCRIPTASE

I N D E R M. V E R M A

Tumor VirologyLaboratory The Salk Institute, Post Office Box 1809, San Diego, Calif. 92112 (U.S.A.) (Received September 28th, 1976)

CONTENTS I.

Introduction

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1.

Life cycle of R N A t u m o u r viruses

1II.

Purification of reverse transcriptase

. . . . . . . . . . . . . . . . . . . . . . .

2

. . . . . . . . . . . . . . . . . . . . . .

4

. . . . . . . . . . . . . . . . . . . . . . . .

4

. . . . . . . . . . . . . . . . . . . . . . . . .

4

A. Sequential c h r o m a t o g r a p h y B. Affinity c h r o m a t o g r a p h y

C. Velocity centrifugation and gel filtration IV.

B. Ribonuclease H

V.

. . . . . . . . . . . . . . . . . .

Enzymatic activities associated with purified reverse transcriptase A. D N A polymerase

2

. . . . . . . . .

4 5

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

C. Other enzymatic activities associated with purified virions or purified reverse transcriptase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Structure of reverse transcriptase

8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 8 10 11 11 12 14 14 14 15 15

. . . . . . . . . . . . . . .

16

A. Avian systems . . . . . . . . . . . . . . . . . . . . . 1. Molecular size . . . . . . . . . . . . . . . . . . . 2. Structural relationship of subunits . . . . . . . . . . . 3. Isolation of a subunit . . . . . . . . . . . . . . . . 4. In vitro conversion of aft to a . . . . . . . . . . . . . 5. Search for fl . . . . . . . . . . . . . . . . . . . . 6. C o m p a r i s o n of properties Of a with aft . . . . . . . . . i. Binding to polymers . . . . . . . . . . . . . . . ii. Thermal inactivation properties . . . . . . . . . . iii. Binding to t R N A primer . . . . . . . . . . . . . iv. Mode of action of R N A a s e H and D N A polymerase B. Reticuloendotheliosis virus reverse transcriptase C. Murine systems

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D. H a m s t e r leukemia virus reverse transcriptase

. . . . . . . . . . . . . . . .

16 16

E. C o m p a r i s o n of properties of reverse transcriptases from avian and murine R N A t u m o r viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

VI.

Immunological properties of reverse transcriptase

. . . . . . . . . . . . . . . .

18

VII.

lnhibitors of reverse transcriptase A. Binding to enzyme . . . . . B. Template binding agents . . . C. Template-primer analogues .

. . . .

19 19 20 20

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

2 VIII. Reverse transcriptase is coded for by the viral genome IX.

The case of L A 6 7 2

X.

RSVa(-)

XI.

Mouse sarcoma viruses

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. . . . . . . . . . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XII. lntracellular reverse transcriptases . . . . . . . . . . . A. Reverse transcriptase in infected cells producing virus B, Reverse transcriptase in nonproducer-infected cells . C. Reverse transcriptase in normal cells . . . . . . . . D. Reverse transcriptase in tumor cells . . . . . . . .

. . . . .

. . . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 .

23 24 24 25 25 26 27 28

XIII. Diagnostic features of reverse transcriptase

. . . . . . . . . . . . . . . . . . .

29

XIV. Reverse transcription . . . . . . . . . . . A. Viral RNA as template . . . . . . . . B, Natural RNAs as tenplate~ . . . . . . C. Transcription of RNAs lacking poly(A)

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30 30 30 32

XV. Future research trends on reverse transcriptase . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32 33 34

. . . .

. . . .

. . . .

I. INTRODUCTION In the s u m m e r o f 1970, Baltimore [1] a n d Temin a n d M i z u t a n i [2] r e p o r t e d the presence o f a D N A p o l y m e r a s e in the virions o f R N A t u m o r viruses. The viral R N A acted as the t e m p l a t e to direct the i n c o r p o r a t i o n o f d e o x y r i b o n u c l e o s i d e triphosphates. This finding furnished credible s u p p o r t to the n o t i o n that R N A t u m o r viruses replicate via a D N A i n t e r m e d i a t e [3,4]. Since its discovery, this enzyme has been an object o f great interest to m a n y investigators and a f o r m i d a b l e a m o u n t o f d a t a has been a c c u m u l a t e d on its propelties, function a n d structure. The enzyme has been alternatively referred to in literature as R N A - d e p e n d e n t D N A polymerase, R N A - d i l e c t e d D N A p o l y m e r a s e and D N A p o l y m e r a s e o f R N A t u m o r viruses (EC 2.7.7.7). In this article, I will refer to it by its m o r e p o p u l a r name, reverse transcriptase. Several o u t s t a n d i n g review articles have recently been written on reverse transcriptase (for genelal i n t r o d u c t i o n , Temin a n d Baltimore [5], T e m i n [6,7], T o o z e [8]; for detailed literature review Sarin a n d G a l l o [9], G r e e n a n d G e r a r d [10], W u a n d G a l l o [11] and S a r n g a d h a r a n et al. [12]). In this article, I shall confine myself to the discussion o f the structure o f reverse transcriptase, its role in the life cycle o f R N A t u m o r viruses a n d b r o a d e r implications to m o l e c u l a r biology. A c c o m p a n y i n g articles b y Taylor, J. a n d Weinberg, R. A. describe in detail the m e c h a n i s m a n d the nature o f D N A synthesized by reverse transcriptase. 1I. LIFE CYCLE OF RNA TUMOR VIRUSES D u r i n g the life cycle o f R N A t u m o r viruses (Fig. 1), early after infection the

cell "~- membrane

nucleus

®'_

vRNA

_

_

/

5'~3'

AA-OH

rxJ"L~AA-0H PROTEINS

I- Penetration

:)-Reverse transcription 3-Provirus transport to nucleus 4 - Integra tion S- Transcription

6-Processing and tram, port 7- Translation 8- Virion assembly 9- Virion rnoturotion

Fig. 1. Life cycle of RNA tumor viruses.

viral RNA is transcribed into double-stranded DNA, probably through an RNADNA hybrid as an intermediate. Covalently closed cizcular DNA (proviral DNA) has been identified in the cytoplasm of the cells infected with either avian sarcoma viruses (ASV) [13] or murine leukemia viruses (MuLV) [14]. The proviral DNA is then transported to the nucleus, where it is integrated into the host genome by some as yet unknown mechanism [I 3]. Subsequent transcription of the integrated proviral DNA (provirus) is probably carried out by cellular DNA-dependent RNA polymerase [15-17]. Neither the nature of the primary transcript of the provirus, nor the mechanism of its processing, if any, is understood. Viral m R N A containing poly(A) has been detected in the cytoplasm [18-21]. Whether aU of the transcribed viral RNA present in the cytoplasm can act as mRNA is not known. The 35 S viral RNA is encapsidated by virus-specific proteins and the maturing virions move to the cell surface, from which they bud, acquiring an outer envelope [8]. Further steps of maturation may occur in the extracellular particles. Reverse transcriptase appears to be required very early after infection to synthesize proviral DNA since viral mutants blocked in this function are unable to establish infection [22]. In order to study the structure and properties of reverse transcriptase of RNA tumor viruses, it is essential to purify it from other viral proteins. Most investigators have isolated reverse transcriptase from virions of either avian or murine RNA tumor viruses. I shall first describe the properties of purified reverse transcriptase from these two sources.

IlL PURIFICATION OF REVERSE TRANSCRIPTASE Reverse transcriptase resides in the core of the virion [8,23]. It can be easily solubilized by lysing the virus with non-ionic detergent like Nonidet P-40 [24-27] or Triton X-100 [28-30], and has been isolated from purified virions by a variety of methods (for details of each procedure, see ref. I l). Generally three main methods have been employed.

IliA. Sequential chromatography The lysed virions are adsorbed either on a DEAE-cellulose or a DEAE-Sephadex column, and the enzyme eluted with a salt gradient. The enzyme is further chromatographed on a phosphocellulose column and enzymatic activity eluted with a salt gradient. Typically, reverse transcriptase elutes from a DEAE-Sephadex column at a salt concentration of 0.06 M to 0.11 M KC1 and from phosphocellulose at 0.2 to 0.3 M KCI [25]. In most instances, purified reverse transcriptase obtained either by first or second phosphocellulose chromatography is essentially free of any other viral protein [31]. The recovery of enzymatic activity from starting material is about 20-40~ by this procedure. On occasion, we have found small amounts of major core protein (p30 in MuLV and p27 in avian viruses) in the purified enzyme [321.

IIIB. Affinity chromatography The strong affinity of reverse transcriptase for various polynucleotides has been exploited by many investigators to purify it from other virion proteins. Columns containing DNA-cellulose [24,33] or oligo(dT) bound to cellulose [34], poly(A)cellulose [35], poly(U)-Sepharose (ref. 36, and Hizi, A. personal communication) and poly(C)-agarose [37] have been used to purify reverse transcriptase from lysed virions. The enzyme is eluted from the column with a salt gradient. Reverse transcriptase obtained from a one-step affinity column is not as pure as the enzyme obtained by sequential chromatography. This one-step purification procedure, is, however, very useful to purify thermolabile reverse transcriptase from temperaturesensitive mutants [38]. Recently, reverse transcriptase has also been purified by affinity chromatography on pyran-Sepharose columns [39]. Pyran copolymer, a divinyl ether of maleic anhydride is not digested by nucleases and thus can be used to purify reverse transcriptase from crude cellular extracts.

lllC. Velocity centrifugation and gel filtration Reverse transcriptase has also been purified by velocity centrifugation in glycerol gradients and by gel filtration. Usually this method has been employed as a final step of purification to eliminate lower molecular weight proteins from the partially purified enzyme. Reverse transcriptase has a tendency to aggregate if centrifugations are carried out in either a low salt or high salt buffer [11 ]. In our experience, a combination of all three methods provides pure enzyme in

high yields. In a typical experiment, the lysed virions are centrifuged at 15 000 × g for 30 min and the supernatant adsorbed onto a poly(C)-agarose column and the. enzyme eluted with a salt gradient. Peak fractions of enzymatic activity are pooled and chromatographed on a phosphocellulose column. Material eluting from phosphocellulose columns is further purified by centrifugation on a glycerol gradient. The yield of enzymatic activity from starting material is 40-50 ~o. The enzyme should be stored at -70°C in 15~ glycerol or at --20°C in 5 0 ~ glycerol. IV. ENZYMATIC ACTIVITIES ASSOCIATED WITH PURIFIED REVERSE TRANSCRIPTASE Purified reverse transcriptase from both avian and murine RNA tumor viruses exhibits both a synthetic and a degradative activity [5,8,10-12]. The synthetic activity is characterized by DNA polymerase activity, whereas the degradative activity is characterized by ribonuclease H (RNAase H) activity.

IliA. DNA polymerase Purified reverse transcriptase can utilize both polyribonucleotides and polydeoxyribonucleotides as templates to direct the synthesis of complementary polydeoxyribonucleotides [5,40,41]. These enzymatic activities have been referred to as RNA-directed DNA polymerase and DNA-directed DNA polymerase. The two activities are inseparable and appear to share a common active site. Both activities have very similar heat inactivation profiles [42]. The enzyme can efficiently and faithfully transcribe both homopolymers and heteropolymers [5,32] (Table I). Like other known DNA polymerases, this enzyme also requires a preformed primer to initiate DNA synthesis [41]. Both ribo and deoxyribo-oligomers can act as primer but deoxyribo-oligomers appear to be more efficient primers [204]. For homopolymeric templates, the primer used is often a complementary deoxyribo-oligomer. For instance, if poly(A) is the template, only an oligomer of T can act as primer [41]. In the virions, the primer for the transcription of 60-70 S viral RNA has been shown to be transfer RNA [43-46]. The primer can be as short as 4-8 nucleotides [41]. The primer presumably provides the 3'-OH end to form a phosphodiester bond with the substrate. The direction of synthesis is from 5' to 3' [47]. The efficiency of transcription of a given template differs with the source of reverse transcriptase and the divalent cations used in the reaction. Table II shows the effect of Mg 2+ and Mn 2+ on the transcription of ribo and deoxyribo homopolymers by purified reverse transcriptase from avian myeloblastosis virus (AMV) or murine luekemia virus. It has been reported that reverse transcriptase from AMV catalyzes the incorporation of an exceptionally large number of incorrectly paired bases when copying ribopolynucleotide and deoxyribopolynucleotide templates [48,49]. The frequency of error is one in 600 when transcribing homopolymer templates and one in 6000 when copying alternating copolymer templates. In contrast, the rate of mis-

TABLE I TRANSCRIPTION OF RIBOPOLYMERS AND DEOXYRIBOPOLYMERS The standard polymerase reaction mixture was used with appropriate template primer, substrate and divalent cations [32]. Template" primer

3H-labeled substrate

Poly(A).oligo(dT) Poly(C).oligo(dG) 70 S AMV RNA with Mg 2+ 70 S AMV RNA with Mn 2÷ 70 S AMV RNA with Mg 2+ ÷ oligo(dT) 70 S M-MuLV CI. I RNA with Mg 2÷ 70 S M-MuLV CI. I RNA with Mn 2+ 10 S rabbit reticulocyte globin RNA ~ oligo(dT) 10 S rabbit reticulocyte globin RNA oligo(dT) ÷ Actinomycin D 18 S slime mold rRNA ÷ oligo(dC) Poly(dC)'oligo(dG) Poly(dA-dT) Activated DNA Native calf thymus DNA Heat denatured calf thymus DNA

dTTP dGTP dATP dATP dATP dATP dATP dGTP

i n c o r p o r a t i o n with Escherichia coli D N A

dGTP dATP dGTP dTTP dTTP dTTP dTTP

M-MuLV DNA polymerase (pmol) 7 280 4 362 3.5 1.25 36 6.6 1.45 500 300 14 11 890 234 45 13 |1

AMV DNA polymerase (pmol) 7 104 7 000 15.5 60 50 595 406 16 4 500 254 39 5.5 8.0

polymerase is one in l0 s nucleotides

po]ymerized. It has been suggested that the higher frequency o f incorrect base i n c o r p o r a t i o n by A M V reverse transcriptase may be due to the absence o f 3 ' - 5 ' exonuclease activity [50]. E. coli and T¢ D N A polymerase possess a 3 ' - 5 ' exonuclease activity [51,52]. A n y m i s m a t c h e d base pair will be excised by the a c c o m p a n y i n g 3 ' - 5 ' exonuclease. H o w e v e r , this m ay not be the entire explanation because purified m a m m a l i a n D N A polymerases have also no detectable nuclease activity but are able to transcribe

TABLE I1 EFFECT OF DIVALENT IONS ON THE TRANSCRIPTION OF RIBOPOLYMERS AND DEOXYRIBOPOLYMERS BY AMV AND M-MuLV REVERSE TRANSCRIPTASE The details of the reaction conditions have been described [32]. Reverse transcriptase

Template- primer

Optimum Mg 2+ (raM)

Incorporation (pmol)

Optimum Mn 2+ (mM)

Incorporation (pmol)

AMV

poly(A).oligo(dT) poly(C)-oligo(dG) poly(dC).oligo(dG) poly(A).oligo(dT) poly(C).oligo(dG) poly(dC).oligo(dG)

6 6 6 2 10 10

20 71 23 74 219 420

1.0 0.1 0.1 1.0 2.0 2.0

7.7 10.0 I 1.0 260.0 30.0 100.0

MuLV

7 homopolymeric templates with higher degrees of accuracy [53]. It is not clear if the high rate of misincorporation with AMV reverse transcriptase is also observed if viral 60 to 70 S RNA is used as template. 1VB. Ribonuclease H

Ribonuclease H activity associated with purified reverse transcriptase specifically degrades the RNA moiety of an RNA.DNA hybrid [5,54-57]. The degradation is not dependent on the concurrent synthesis of complementary DNA (cDNA) since preformed hybrids are susceptible to degradation [32,55]. It acts as an exoribonuclease and requires free ends [56]. In contrast, cellular RNAase H is an endoribonuclease [57]. The polarity of the nuclease action has not yet been established. The products of the reaction using [3H]poly(A).poly(dT) as substrate have been identified as a series of oligonucleotides ranging from 6 to 20 adenylate residues [32,55,58]. No AMP is detected among the reaction products. Ribonuclease H associated with purified reverse transcriptase from either AMV or MuLV cleaves at the 3' end of the 3'-5' phosphodiester bound to yield products containing 5' phosphate and 3'-OH ends [32,55,56]. RNAase H associated with AMV reverse transcriptase acts as a processive exonuclease [32,59], whereas RNAase H associated with MuLV DNA polymerase acts predominantly as a random exonuclease [32]. A processive nuclease is defined as an enzyme which once bound to the polynucleotide chain substrate completely degrades the substrate before being released. In a recent publication, it has been shown that RNAase H activity associated with purified reverse transcriptase from Friend-MuLV is a processive exonuclease. However, upon storage the enzyme degrades and acts as random exonuclease [58]. The nature and mode of action of RNAase H activity associated with MuLV reverse transcriptase thus remains unclear. It appears from all available data that although RNAase H and DNA polymerase activities reside on the same polypeptide, they have different functional sites. The following data support this notion: (a) DNA polymerase activity is more heat labile than the corresponding RNAase H activity [42,56]. (b) RNAase H activity is selectively inhibited by NaF. At 30 mM NaF and 150 mM KCI, over 80% of the RNAase H activity is inhibited, with little effect on reverse transcriptase activity [60]. (c) Digestion of reverse transcriptase with chymotrypsin leads to inactivation of DNA polymerase activity 8- to 10-times faster than the corresponding RNAase H activity (Lai, M. T., personal communication). (d) DNA polymerase activity purified from AMV is inactivated 8-times faster than the RNAase H activity by N-ethylmaleJmide (Panet, A., personal communication). (e) RNAase H activity associated with purified reverse transcriptase is more sensitive to inhibiton by poly(dA) than the DNA polymerase activity (Panet, A., personal communication). The role of RNAase H activity during the process of reverse transcription remains unclear. The digested RNA product has a pS' and 3'-OH, suggesting that the degraded RNA template can act as primer for the synthesis of the second strand

of DNA. However, double-stranded DNA can be synthesized when experiments are performed in the presence of NaF, an inhibitor of RNAase H activity [32,61]. IVC. Other enzymatic activities associated with purified virions or purified reverse transcriptase

Several enzymatic activities have been reported to be present in the virions of RNA tumor viruses. These include deoxyribonuclease [28,62], protein kinase [64], nucleotide kinase [65], phosphatase [65], hexokinase [65], lactic dehydrogenase [65], ribonuclease [66] and DNA ligase [67]. None of these activities has been well characterized. Some of these activities are almost certainly not coded for by the viral RNA, but may play a role in the process of reverse transcription. Recently it was reported that partially purified reverse transcriptase from AMV also exhibits nucleoside diphosphokinase activity [68]. It will be interesting to see if this activity is an integral part of the reverse transcriptase. 1 think it is fair to conclude that at the present time, only DNA polymerase and RNAase H activities can be unambiguously attributed to the RNA tumor viruses.

v. STRUCTURE OF REVERSE TRANSCRIPTASE The structme of purified reverse transcriptase isolated from avian and murine RNA tumor viruses is quite different. I shall therefore discuss the two systems separately. VA. Avian system 1. Molecular size. Purified reverse transcriptase from avian RNA tumor

viruses, upon centrifugation in glycerol gradients, sediments at 7.5 S [5,11,26,31,63] as compared to the sedimentation coefficient of 5.6 S [69] for E. coli DNA polymerase I. By using the formula (mwl)2/3/(mw2) =- $1/$2 [70], the size of the reverse transcriptase from avian RNA tumor viruses appears to be 170 000 daltons. It is excluded in the void volume, when chromatographed on a G-100 Sephadex column [27,71]. However, when the enzyme is analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gels, two polypeptides of an apparent molecular weight of 95 000 (t3) and 65 000 (a) are evident [24,26,72]. The two polypeptides are in equimolar ratio and the molecular weights of the two subunits add up to the size of the undissociated enzyme. The reverse transcriptase from avian RNA tumor viruses thus appears to be a two-subunit complex. Upon prolonged storage of purified enzyme at either -20 or -70 °C, the quantity of large subunit/3 is decreased with concomitant increase in the ratio and amount of the small subunit a and appearance of several smaller polypeptides of molecular weights ranging fiom 20 000 to 40 000 (ref. 73 and Lai, M. T. and Verma, I. M., unpublished results). It is not clear whether the prolonged storage also affects the integrity of small subunit a. if the enzyme is stored at high protein concentrations, much less fl appears to be converted to a and smaller poly-

9

A

B

C

D

E.

Fig. 2. Effect of prolonged storage on the physical integrity of purified AMV reverse transcriptase. Polyacrylamide gel electrophoresis of freshly purified (2 weeks old) and 14 months old (stored at --90°C in 15 ~ glycerol) AMV reverse transcriptase. (A) molecular weights of standards, fl-galactosidase, 130 000; bovine serum albumin 68 000; ovalbumin, 46 000; chymotrypsinogen, 25 700; and cytochrome c, 12 000. (B) Fresh enzyme stored at a concentration of 50/~g/ml. (C) Old enzyme stored at a concentration of 14/~g/ml. (D) Old enzyme stored at a concentration of 150/~g/ml. (E) Old enzyme stored at a concentration of 37 f,g/ml.

peptides (see Fig. 2). It appears that this conversion offl to a and smaller polypeptides may be the result of proteolytic activity in the storage buffer. It has recently been reported that at least one of the components of storage buffer, glycerol, contains proteolytic activity [74]. Presence of a protease inhibitor phenazine methosulfate fluoride (PMSF) during purification of the enzyme leads to higher ratios of fl to a [73]. Enzymatic preparations with ratios of/3 to a less than 1 are generally less active than the preparations containing fl to a in equimolar amounts [73]. On the other hand, preparations of enzyme with ratios of ~8:a= 1.9 have been shown to be more potent than enzymatic preparations containing fl to a in molar ratios of 1 [73]. 2. Structural relationship of subunits. The decrease in the amount of fl with concomitant increase in the amount of a suggests that the two subunits may be structurally related. Direct proof of this notion was obtained by comparing the peptide maps of isolated a and fl subunits by two-dimensional finger-printing analyses [72]. Fig. 3 shows the peptide maps of a, fl and a mixture of a and ft. It can be seen that the general distribution of radioactive spots is strikingly similar in all cases. With the possible exception of several 12sI-labeled peptides in the region of spots n and o, all the 12SI-labeled peptides of the small subunit are also present in the large subunit. If a subunit is derived from fl by cleavage at the NH2 terminal, or C D O H terminal, or at both ends, one or two new peptides could be generated. The peptide maps of isolated a and fl subunits from RSV wele also shown to be structurally related [72]. Furthermore, the two-dimensional tryptic peptide distributions of the reverse transcriptase subunits of AMV and RSV resemble each other closely, suggest-

10

/I

8

a"

--k

IN-..

Fig. 3. Tryptic-peptides analyses of the a and {~ subunits of purified AMV reverse transcriptase. Purified reverse transcriptase from AMV was radiolabeled with t251 and the two subunits isolated on SDS polyacrylamide gels. The protein bands were located by staining with dye (Comassie brilliant blue) and autoradiography. The ratio of fl:a was not significantly altered by iodination procedures. 12Si.labeled a and fl subunits were excised from the gel and digested with trypsin. These photographs show the autoradiograms prepared from two-dimensional separations of tryptic hydrolysates. A. a subunit; B. fl subunit; and C. a mixture of a ÷ ~. For details see ref. 72. ing that the two enzymes have similar primary structure. This is consistent with the observation that antisera to reverse transcriptase o f A M Y inhibit the enzymatic activity of reverse transcriptase of RSV [75]. These observations have been confirmed and extended by Rho et al. [76] who have compared the tryptic and chymotryptic maps of z2 ~l-labeled a and fl subunits o f A M V reverse transcriptase. Furthermore, the peptide maps o f free a subunits and n subunits obtained from aft complexes were shown to be identical. It thus appears that a and fl are structurally related and that a is perhaps derived from ft. I f the small subunit a is derived from the larger precursor/~ and the most a b u n d a n t and stable form of the enzyme is an aft complex, then reverse transcriptase of avian R N A t u m o r viruses is an unique example of an enzyme that requires a close precursorproduct association for maximal enzymatic acitivity. 3. Isolation ofa subunit. Grandgenett et al. [20] were able to isolate a subunit from the lysed virions by chromatography on phosphocellulose columns. Isolated a has an average molecular weight of 65 000, as tested by three independent methods: SDS polyacrylamide gel electrophoresis, velocity sedimentation on glycerol gradients, and gel filtration [26,71]. The a subunit manifests both D N A polymerase and R N A a s e H activities. The ratios of specific activities of isolated a and aft complexes (holoenzyme) in response to several R N A and D N A polymers tested as templates remained essentially constant [26]. Antibodies raised against purified holoenzyme can crossreact with both isolated a and aft forms o f enzyme [33,77]. The a subunit can be generated from purified aft complex by a variety of procedures and isolated by c h r o m a t o g r a p h y on a phosphocellulose column. The a subunit elutes from phosphocellulose at lower salt (0.1 M K C I ) compared to aft complex (0.3 M KC1) [26,71].

II

A

B

C

D

E

--95K 68 K--

-- 39K --30K --22K

Fig. 4. Polyacrylamide gel electrophoretic analyses of chymotrypsin-treated AMV reverse transcriptase. Purified AMV reverse transcriptase was digested with chymotrypsin (5 /~g/ml) at 37°C. The reaction was stopped by addition of protease inhibitor, PMSF. Aliquots were withdrawn for reverse transcriptase assays and the remainder of the sample was analyzed. (A) Molecular weight of standards, fl galactosidase, 130 000; bovine serum albumin 68 000; ovalbumin, 46 000; chymotrypsinogen, 25 700; and cytochrome c, 12 000. (B) Undigested enzyme. (C) 15 min digestion. (D) 30 min digestion. (E) Chymotrypsin.

4. In vitro conversion of aft to a. The holoenzyme can be converted in vitro to a either by proteolytic enzymes (ref. 73 and Lai, M. T. and Verma, I. M., unpublished results) or organic chemicals [78]. Fig. 4 shows gel electropho]etic patterns o f holoenzyme incubated with 5 ~zg/ml o f chymotrypsin for different periods o f time at 37°C. It can be observed that in 15 rain over 60-70~o of fl subunit is converted into a polypeptide indistinguishable from a subunit. Furthermore, several smaller polypeptides with molecular weights ranging from 20 000 to 40 000 are also evident. This pattern of conversion o f aft to a and smaller polypeptides is quite similar to the pattern obtained f r o m enzyme stored for prolonged periods of time. U n d e r the conditions, where a majority of fl subunit in aft complexes is converted to a and smaller

12 polypeptides, little loss of enzymatic activity was observed (Lai, M.T. and Verma, I.M., unpublished results). From these observations, however, it is not clear whether some a is also digested into smaller polypeptides. Recently Grandgenett has reported that dimethyl sulfoxide (30~o v/v)[78] or dioxane (15 ~ v/v)[79] can dissociate a13 complex to yield enzymatically active subunits. Chromatography of dimethyl sulfoxide-treated aft on a phosphocellulose column shows the appearance of three enzymatic peaks corresponding in size to a, a/3 and a fraction enriched in/3. It thus appears that the isolated a/3 complex can be converted in vitro to an active a subunit form. 5. Search for /3. Attempts to isolate t3 subunit in native form have not been very successful. Perhaps 13 subunit is very labile and is rapidly converted to a and smaller polypeptides. Furthermore, it is also possible that the/3 subunit of reverse transcriptase is not enzymatically active and hence cannot be assayed by conventional methods. Keeping this in mind, we radiolabeled purified reverse transcriptase with ~2Sl by utilizing lactoperoxidase bound to Sepharose to mediate the iodination reaction [80]. This procedure allowed us to obtain 1251-labeled reverse transcriptase with full enzymatic activity. The enzyme was then dialyzed overnight against buffer to generate a subunit and fractionated on a phosphocellulose column with a salt gradient of 0.1 to 0.6 M KCI. Both the enzymatic activity and ~25I radioactivity were monitored (Fig. 5). Fractions indicated in the figure were analyzed on an SDS polyacrylamide gel. It can be seen that the two peaks of enzymatic activity eluting at a salt concentration of 0.11 M KC 1 and 0.24 M KC 1 upon analysis on polyacrylamide gel (fraction Nos. 31 and 61) show typical patterns of a and aft. A new fragment of molecular weight approx. 23 000 can also be observed in the slot showing a subunit. The nature of this fragment is under investigation. However, radioactive material is spread all along the salt gradient. Analysis of radioactive material from various regions of the column on an SDS polyacrylamide gel (fractions 43, 50 and 54) showed mostly a with trace amounts of t3 subunit. We have no explanation as to why these samples are not enzymatically active. Neither enzymatic activity nor radioactivity corresponding to isolated/3 subunit could be detected. It is possible that/3 subunit binds tightly to the phosphocellulose column and elutes at much higher salt concentrations. So far we have not been able to elute any enzymatic activity or radioactive material from the column, even with 2 M KCI. Hizi and Joklik have recently reported (personal communication) the isolation of t3t3 dimer from virions of B77 strain of avian sarcoma viruses grown in duck embryo fibroblasts. By using sequential chromatography on DEAE cellulose, followed by phosphocellulose and poly(U)-Sepharose, they were able to isolate enzymatically active 13/3 form of the enzyme. The isolated 13fl dimer has a molecular weight of 170 000 and sediments at 7.7 S in glycerol gradients. Isolated 13t3, upon analysis on SDS polyacrylamide gel, shows only one polypeptide with average molecular weight of 85 000. Isolated a, a/3 and 1313 forms of enzyme exhibit identical enzymatic properties. However, transcription of genomic 70 S RNA is carried out most efficiently with a13 form of the enzyme. It is intriguing that these investigators have

13

O,IIM KCI

0 . 2 4 M KCI

0.6 M KCI

2OO 140

,I00 x

/

.

~

\.,q 50 8_

w,

,2[ /

S

~--e--~,. 0

-"t

i

2b

35

ao ~

49

s5

8o

!

--9~5K -

Fig. 5. Column chromatography and gel electrophoretic analysis of 12SI-labeled AMV reverse transcriptase. ~25I-labeled enzyme was dialyzed overnight and chromatographed on a phosphocellulose column. The enzymatic activity was monitored by assaying RNA-dependent DNA polymerase activity with poly(C).oligo(dG) as described [25] and radioactivity determined in a gamma counter. Samples from indicated fractions were analyzed by polyacrylamide gel electropheresis. Over 90~ of enzymic activity was recovered. The molecular weights of fl, a and new fragment in fraction No. 35 are indicated.

succeeded in isolating the tiff form of the enzyme where others have failed, using similar conditions. It may be that the tiff form of enzyme in B77 grown in duck fibroblasts is more stable than the reverse transcriptase isolated from A M V or RSV. The partial physical map of the genome of avian sarcoma viruses has been constructed and it ,appears that the maximum coding capacity available for the synthesis of reverse transcriptase is about 3000 nucleotides corresponding to 100 000 dalton proteins [22]. The available data, however, do not rule out the rather unlikely possibility that the two subunits are coded for by separate but related genes. The following simple scheme of events leading to the synthesis of most stable form of enzyme, aft complex, appears most likely. The initial intracellular form of reverse transcriptase is a tiff dimer. One of the two subunits of the dimer is specifically cleaved, presumably by a proteolytic enzyme, to give rise to aft complexes. The configuration of aft dimer affords enhanced resistance to further cleavage with proteolytic enzymes. The a subunit is perhaps a decay product of aft. It is not clear whether an aa dimer is generated during the conversion of aft dimer to a and smaller

14 polypeptides. The isolation of tiff dimer by Hizi and Joklik should make it feasible to test the scheme

ff-->

af-->

a

6. Comparison of properties of a with aft. Both a and a f manifest D N A polymerase and R N A a s e H activities [26]. However, the two forms o f reverse transcriptase exhibit markedly different extents of binding to templates [33,35]. Furthermore, the mode of action of D N A polymerase and R N A a s e H activities associated with a and a f is different [59]. 6i. Binding to polymers. Purified a subunit has lower binding affinity to polymers than the holoenzyme, af. Both a and aft can bind to a DNA-cellulose column, but a can be eluted from the column at a salt concentration of 0.14 M K C I , whereas a f eluted from the column at a salt concentration of 0.25 M K C I [33]. Grandgenett and R h o [35] have shown that while purified a is unable to bind to oligo(dT)-cellulose, poly(A)-cellulose or poly(C)-Sepharose, a f binds quite tightly to these polymers. Thus it appears that a is deficient in its ability to bind to polymers as compared to aft. 6ii. Thermal inactivation properties. In m a n y enzymatic systems it has been shown that specific substrates are able to protect enzymes from inactivation by various factors and the extent o f protection depends on the binding affinity o f the enzyme for the substrate [81]. The heat inactivation profiles of the two forms of the viral enzyme are quite similar [32]. If, however, the enzyme is heat inactivated in a reaction mixture containing template, the enzymatic activity of a f is partially protected, but the a subunit is not at all protected [33]. The template is unable to bind to a subunit to protect it from thermal inactivation. As mentioned before, purified reverse transcriptase from A M V stored at - 9 0 ° C for over a 14-month period, upon analysis, is found to be devoid of t3. Instead, it contains a subunit as its prominent c o m p o n e n t and two smaller polypeptides with the apparent molecular weights o f 38 000 and 25 000. Thermal inactivation properties of old enzyme, when compared TABLE III EFFECT OF PRESENCE OF TEMPLATE ON THE HEAT INACTIVATION PROPERTIES OF aft, a AND OLD ENZYME Reverse transcriptase was incubated at 46 °C in the presence or absence of'poly(A). Aliquots were withdrawn at various time intervals and assayed for DNA polyrnerase activity. The old enzyme used was stored at a concentration of 14 pg/ml (Fig. 2, panel C). t1~2 is the time required for inactivation of 50 ~ of activity at indicated temperature. Source

aft

Presence of template • primer in the reaction mix -

-

+ a

- -

Old enzyme

_L -+

(tv2) at 46°C (rain) 7.0 15.5 7.5 7.5 6.5 14.5

15 to those of aft, are different from those of isolated a. Table III gives the time required for inactivation of 50 ~ of activity (tl/2) of aft, a and old enzyme, when assayed with poly(A).oligo(dT) as template.primer. It thus appears that the smaller polypeptides originating upon conversion of aft to a may be involved in the binding of template. We are now isolating these smaller polypeptides to compare their peptide maps with isolated a and fl subunits. 6iii. Binding to tRNA primer. Reverse transcriptase requires an RNA primer to initiate DNA synthesis directed by 60-70 S viral RNA. For Rous sarcoma virus, the RNA primer required to initiate cDNA synthesis has been shown to be identical to a cellular tryptophan tRNA (tRNA T'~) [45]. Panet et al. [72] have shown that tRNA T'p isolated from either virions of RSV or cellular extracts has strong and specific binding affinity to purified reverse transcriptase from AMV. Recently, Grandgenett et al. [83] have demonstrated that isolated a subunit, however, does not bind to tRNA Trp, even at a 1000-fold molar excess. The aft and a form of enzyme enriched in fl subunit (peak III) bind with high affinity to tRNA rrp. Baltimore and his associates (personal communication) have also shown that the primer tRNA Trp has a much lower binding affinity to a subunit as compared to a/~ complex. The a subunit thus appears to have lower binding affinity for both the template and tRNA Trp primer. The significance of specific binding of primer to the enzyme is still obscure. Isolated a can apparently utilize 70 S viral RNA as template to synthesize complementary DNA. It is possible that trace amounts of residual fl in preparations of isolated a can provide the proper enzyme.primer complex. It will be interesting to find whether the old enzyme can bind to tRNA Trp. 6iv. Mode of action of RNAase H and DNA polymerase. The holoenzyme aft acts as a processive exoribonuclease, since it degrades one polynucleotide chain completely prior to initiating hydrolysis on the second chain. In contrast, a subunit exhibits a random exoribonuclease activity which results in the release of the substrate molecule after each chain scission [59]. Inability of a to bind tightly to the substrate appears to be responsible for the random scission of substrate molecules. The old enzyme containing a and two smaller polypeptides, however, acted as a processive ribonuclease H (Lai, M. T. and Verma, I. M., unpublished results). Leis, J. and Smith, R. G. (personal communication) have observed that aft form of purified AMV and RSV reverse transcriptase acts processively in catalyzing DNA synthesis primed with heteropolymers and homopolymers. In contrast, isolated a subunit acts nonprocessively. They have also observed that DNA polymerase activity associated with the tiff form of reverse transcriptase (isolated by Hizi and Joklik from B77 virus) acts processively. Thus the major difference between the two best studied forms of reverse transcriptase, a and aft, appears to be the lack of binding affinity of a to polynucleotides. The ability of old enzyme containing primarily a and two smaller polypeptides to mimic the binding properties of a~ form suggests that the polypeptides cleaved from/~ to generate a are involved in binding to polynucleotides.

16 VB. Reticuloendotheliosis virus reverse transcriptase

Reticuloendotheliosis virus (REV) is the prototype of a newly recognized group of avian RNA tumor viruses [84], unrelated to the avian leukemia sarcoma virus (ALSV) complex. This group includes spleen necrosis virus (SNV), REV (strain T), duck infectious anemia virus, and chick syncytial virus [84]. Like the other RNA tumor viruses, they possess reverse transcriptase and their replication proceeds via a DNA provirus integrated with host cell DNA [85,86]. It was earlier reported that REV was incapable of exhibiting endogenous DNA polymerase activity [87]. It now appears that if Mn 2+ is used as the divalent ion instead of Mg 2+, sufficient endogenous activity can be demonstrated [88,90]. The REV DNA polymerases are closely related to each other by serological tests; however, they do not share detectable homology with the reverse transcriptase isolated from ALSV [91]. Purified reverse transcriptase from REV exhibits both DNA polymerase and RNAase H activities [92]. Unlike reverse transcriptases from ALSV, purified reverse transcriptase from REV appears to contain only one polypeptide chain of molecular weight of 70 000 [92] to 84 000 [901. VC. Murine systems

Purified reverse transcriptase from murine leukemia viruses sediments on a glycerol gradient at 4-4.5 S [11,32,93]. It manifests both the DNA polymerase and RNAase H activities [32,73,93]. Only one polypeptide of a molecular weight ranging from 70 000 to 84 000 has been found [11,32,93]. Gerard and Grandgenett [94] have reported that electrophoresis of purified reverse transcriptase from Moloney murine sarcoma-leukemia virus on SDS polyacrylamide gels show three polypeptides of molecular weights of 82 000, 68 000 and 60 000. Unlike the a and t3 subunits of reverse transcriptase from avian RNA tumor viruses, these three polypeptides do not appear to be in equimolar ratio. Though the reverse transcfiptase was isolated from a murine sarcoma-leukemia complex, it is probably coded for by the leukemia virus genome because the murine sarcoma viruses appear to be defective in the synthesis of reverse transcriptase [95,96]. It has recently been reported that purified reverse transt, riptase from Friend murine leukemia virus is rapidly degraded upon storage [58]. It seems likely that the 68 000 and 60 000 dalton polypeptides present in purified Moloney sarcoma leukemia virus represent degradation products of the 82 000 polypeptide. VD. Hamster leukemia virus reverse transcriptase

Purified reverse transcriptase from another mammalian virus, hamster leukemia virus (HaLV), appears to have a molecular weight of 120 000 and contains two polypeptides with average molecular weights of 68 000 and 53 000 [31]. It was originally reported that purified HaLV reverse transcriptase has no demonstrable RNAase H activity [31]. With more sensitive substrates, we have now been able to detect RNAase H activity associated with purified HaLV reverse transcriptase (Verma, I. M., unpublished results).

17 TABLE IV COMPARISON OF PROPERTIES OF M-MuLV REVERSE TRANSCRIPTASE, ISOLATED a AND aft FORMS OF AMV REVERSE TRANSCRIPTASE Properties

MuLV

a

aft

Molecular weight Number of subunits tl/2 of DNA polymerase (in rain, at 45°C) tl/2 of DNA polymerase with template (in rain at 45°C) Binding affinity of primer tRNA Mode of action of RNAase H

84 000 1 6,5 6.5 very low random

65 000 l 7.5 7.5 very low random

170 000 2 7.0 15.5 very high processive

VE. Comparison of properties of reverse transcriptases from avian and murine RNA tumor viruses Isolated a and a/3 forms of reverse transcriptase from AMV differ in their template binding properties and mode of action of RNAase H activity. Are the properties of MuLV reverse transcriptase similar to isolated a or aft forms of A M V reverse transcriptase? Like the a subunit of AMV, purified D N A polymerase from M - M u L V is not protected by template-primer from thermal inactivation [32]. Ribonuclease H activity associated with M - M u L V reverse transcriptase acts as a random exonuclease [32]. Furthermore, purified reverse transcriptase from M - M u L V shows very low binding affinity to the R N A primer, t R N A Pr° (Baltimore, D. personal communication). The properties of purified reverse transcriptase isolated from avian and murine R N A tumor viruses are compared in Table IV. On the basis of its binding properties and mode of action of RNAase H activity, it appears that purified reverse transcriptase from murine R N A tumor viruses is like the a form of A M V reverse transcriptase. It is tempting to speculate that murine reverse transcriptase is also a twosubunit enzyme, but the larger or precursor subunit is very labile. Perhaps the enzyme should be isolated from infected cells instead of released virions. It should also be interesting to study the mechanistic properties of isolated subunit and two-subunit complex reverse transcriptase from HaLV. Contrary to the report [32] that RNAase H activity associated with purified reverse transcriptase from M-MuLV acts as a random exonuclease, it has recently been reported [58] that purified reverse transcriptase from Friend-MuLV is a processive exonuclease. It was shown that Friend-MuLV reverse transcriptase is very labile and rapidly degrades upon storage [58]. The degraded enzyme, however, still retained a large proportion of 84 000 dalton polypeptide. The properties of degraded enzyme were comparable to the properties of isolated a form of A M V reverse transcriptase. However, intact purified reverse transcriptase of an apparent molecular weight of 84 000 displays properties identical to those of aft form of AMV reverse transcriptase. The mode of action of RNAase H associated with purified reverse transcriptase from MuLV appears to be uncertain in view of these two conflicting reports [32,58].

18 Vl. IMMUNOLOGICAL PROPERTIES OF REVERSE TRANSCRIPTASE Antisera against reverse transcriptase can be obtained either from animals bearing tumors or, more often, from animals immunized with partially purified reverse transcriptase [97,98]. Reverse transcriptases from avian RNA tumor viruses exhibit immunological cross-reactivity [91]. Thus, a monospecific antiserum prepared against partially purified reverse transcriptase from AMV strongly inhibited the homologous enzyme activity as well as the polymerase activity in disrupted virions of Schmidt-Ruppin (SR) RSV, B77 RSV, and Rous associated viruses. It did not neutralize the DNA polymerase activities of MuLV, feline leukemia virus (FeLV), Visna virus, mouse mammary tumor virus (MMTV) and Mason-Pfizer mammary tumor virus (M-PMV) [75]. Similarly, antisera laised against RSV reverse transcriptase cross-reacted with reverse transcriptase from only ALSV complex. Panet et al. [77] have developed a radioimmunoassay which can detect and quantitate very small amounts of reverse transcriptase. The assay was unable to detect antigenic sites in either RSVp(-) virions which exhibited no detectable reverse transcriptase by enzymatic assays [99], or in virions of murine RNA tumor viruses. About 70 molecules of reverse transcriptase were found per virion of AMV with radioimmunoassay. Radioimmunoassay should prove very useful to detect a presence of enzymatically inactive protein in the infected or uninfected cells. Monospecific antibodies raised against purified AMV reverse tlanscriptase (holoenzyme) inhibited enzymatic activities associated with both a and aft forms of the enzyme [77]. Antibodies raised against purified AMV can inhibit both the DNA polymerase and RNAase H activities [92]. Although antibodies to reverse transcriptase from mammalian C-type viruses have been reported, no monospecific antiserum is yet available (Fan, H., personal communication; Baltimore, D., personal communication). Antisera raised against partially purified Rauscher MuLV (R-MuLV) reverse transcriptase neutralizes homologous polymerase most strongly, but it also exhibits substantial cross-reactivity with the polymerases of rat leukemia virus, HaLV and FeLV [97]. It has no detectable cross-reactivity with reverse transcriptase from ALSV complex. Neither anti-MuLV nor anti-FeLV sera has any significant neutralizing activity against the reverse trancriptase of two known primate type-C viruses, namely Wooley monkey virus and Gibbon ape virus (GaLV) [100]. Conversely, the antisera raised against either of these viral reverse transcriptases, while strongly neutralizing both of the primate viral enzymes, has no cross-reactivity with feline or murine RNA tumor virus leverse transcriptases [101,102]. Reverse transcriptase of endogenous viruses derived from the same species or related species are often antigenically distinct from the reverse transcriptase of the exogenous viruses infecting the same species. Reverse transcriptases of a group of endogenous cat viruses were immunologically closely related to each other but were quite unrelated to the exogenous virus of the same species FeLV [103].

19 Reverse transcriptase isolated from Viper RNA tumor virus, Visna virus, syncytium forming virus ("foamy" virus), MMTV and M-PMV do not show any cross-reactivity with known anti-revel se transcriptase antibodies from any other virus [97,102]. Conversely, antisera raised against the reverse transcriptase from M-PMV does not inhibit reverse transcriptase of Wooley monkey virus, GaLV, FeLV, R-MuLV, M-MTV and AMV [101,104]. Antisera raised against purified reverse transcriptase from RNA tumor viruses do not cross-react with cellular DNA polymerases [11,91,105]. Antibodies raised against purified cellular DNA polymerase a did not cross-react with either cellular DNA polymerases fl or y or viral reverse transcriptases. The results obtained from antigenic cross-reactivity of reverse transcriptases from various viruses can be summarized as follows: (i) Reverse transcriptases from avian RNA tumor viruses cross-react with each other, the exception being REV reverse transcriptase which did not cross-react with reverse transcriptase from ALSV complex. (ii) Reverse transcriptases from type C mammalian viruses were not detectably related to reverse transcriptase from avian type C viruses. (iii) Reverse transcriptases from RNA tumor viruses of various species of lower mammals (e.g., eats, rats, mice) were related but distinguishable. (iv) Reverse transcriptases from lower mammalian RNA tumor viruses showed limited cross-reactivity with reverse transcriptase from primate RNA tumor viruses. (v) Reverse transcriptases from endogenous type C viruses appeared to be related and were distinct from the exogenous viruses. (vi) Reverse transcriptase from B-type RNA tumor viruses did not cross-react with the reverse transcriptase of C-type RNA tumor viruses. (vii) Reverse transcriptases did not cross-react with cellular DNA polymerases. (viii) Reverse transcriptases isolated from blood cells of patients with A M L were closely related to reverse transcriptase flora primate type C RNA tumor viruses (see Sect. XIID).

VII. INHIBITORS OF REVERSE TRANSCRIPTASE Despite the enormous efforts of several investigators, no specific inhibitor of reverse transcriptase is yet available. Basically, three kinds of inhibitors have been studied, namely, (a) those that bind to reverse transcriptase; (b) those that bind to template, and (c) those that are analogues of template-primer. VIIA. Binding to enzyme

This group includes rifamycin SV derivatives [ 106-109], streptovaricins [110,111 ] and alkaloid extract [112]. Rifamycin SV derivatives are probably the best studied inhibitors of reverse transcriptase. However, no rifamycin derivative which selectively inhibits reverse transcriptase and no cellular DNA-dependent RNA polymerase have yet been reported. The mechanism of action of one derivative, 2,5-dimethyl-4-Nbenzyldemethyl rifampicin (AF/ABDMP) has been studied extensively 014].

20 Inhibition by AF/ABDMP is noncompetitive with respect to template and substrate.

VIIB. Template binding agents This group includes pyran copolymer [113], actinomycin D [115,116], daunomycin, distamycin, ethidium bromide, chromamycin, parsomycin, adriamycin, cinerubin, proflavin, tilurone, acridine orange, congo red, histone and protamine [117-124]. They inhibit DNA synthesis by binding to some bases. VIIC. Template-primer analogues Compounds in this group act by either binding tightly to reverse transcriptase or competing with template-primer for its binding site. The group includes thymidylate derivatives, polyribonucleotides, modified polyribonucleotides, such as thiolated polycytidylate or 2'-o-alkylated polyadenylic acids. The potency of inhibition by polynucleotides is as follows: poly(U) > poly(G) > poly(A) > poly(C) [11,125]. In addition to these three groups of inhibitors, several other agents have been reported to exert inhibitory effects on reverse transcriptase. These include: streptonigrin [126], bleomycin [127], heparin [116], and N-methylisatin-fl-thiosemicarbazone [128]. Substrate analogues like ara-CTP [129] and dideoxythymidine triphosphate [47], can also inhibit reverse transcriptase activity by not allowing chain elongation. Since reverse transcriptase is a zinc metalloenzyme [130,131], it is also inhibited by the chelating agent, orthophenanthroline [130]. Sodium fluoride inhibits RNAase H activity associated with purified reverse transcriptase [60]. N-ethylmaleimide also inhibits DNA polymerase and RNAase H activities of reverse transcriptase, presumably by binding to the reactive SH groups in the active sites [56,75]. VIlI. REVERSE TRANSCRIPTASE 1S CODED FOR BY THE VIRAL GENOME Linial and Mason [132] isolated two temperature-sensitive mutants of RSV (LA335 and LA337) which were unable to transform cells in vitro or replicate at nonpermissive temperature (41 °C). The lesions appear to be expressed after adsorption to and penetration into the host cells. These mutants appeared to be defective in an early function, and exhibited a more thermolabile reverse transcriptase than the parent wild-type. Revertants of LA335 and LA337 [133] which are no longer deiective for infection at 41 °C possess a heat staNe reverse transcriptase activity [142]. Furthermore, recombinants between LA335 and LA337 and an avian leukosis virus [22,133], which have acquired the ability to infect at nonpermissive temperatures, simultaneously acquire a heat stable polymerase activity. Thus it appears that LA335 and LA337 have a lesion in their reverse transcriptase. Reverse transcriptase has been purified from at least three temperature-sensitive mutants (LA335, LA336 and LA337) of Rous sarcoma virus with defects in early functions [38,42,71,134,135]. The heat inactivation properties of DNA polymerase and RNAase H activities have been compared with the corresponding activities from the wild-type parent. Some of the results have been compiled in Table V and can be summarized as follows.

21 TABLE V AVERAGE HALF-TIME (t~/2) OF DNA POLYMERASE AND RNAase H ACTIVITIES OF SEVERAL TEMPERATURE-SENSITIVE MUTANTS OF AVIAN SARCOMA VIRUSES AND THEIR WILD-TYPE PARENTS For details see ref. 38. tl/2 is the time required for inactivation of 50~ of activity at the indicated temperature. Source

Wild-type PR-C RSV LA335 LA337 LA338 Wild-type B 77-C RSV LA336

Temperature (°c)

t1~2 in rain RNA-directed DNA polymerase

DNA-directed DNA polymerase

Ribonuclease H

44 44 44 45 34 34

11.5 2.0 1.8 12.0 30.0 2.6

11.5 3.0 2.8 11.0 30.0 2.7

30.0 7.4 5.0 20.0 50.0 2.0

(i) Both the D N A polymerase and RNAase H activities associated with the purified reverse transcriptases fromLA335, LA336, and LA337, are more heat labile than the corresponding activities of purified reverse transcriptase from the wild-type parent. (ii) D N A polymerase and RNAase H activities associated with the revertants of LA335 and LA337 are no more heat labile than the corresponding enzymatic activities from the wild-type parent. (iii) In the enzymes from all three mutants, both the D N A polymerase and RNAase H activities are more thermolabile than the enzyme obtained from wild-type virus. (iv) Both the D N A polymerase and RNAase H activities associated with isolated a subunit from L,4337 D N A polymerase are more thermolabile than the reverse transcriptase activity associated with purified a subunit of wild-type parent. (v) Preincubation of wild-type enzyme with template shows increased protection from thermal degradation. But the thermosensitivity of purified reverse transcriptase from LA335 and LA337 are not affected if preincubations are carried out in the presence of template-primer. If purified reverse transcriptase from LA336 is preheated with template, marked protection from thermal inactivation is evident. Thus it appears that the mutants, LA335 and LA337, have a lesion distinguishable from that of LA336. All three mutants bearing thermolabile polymerases are deficient in viral D N A synthesis when used to infect duck embryo cells at the nonpermissive temperature, as measured by nucleic acid hybridization [13,38]. Viral D N A synthesis in cells infected by wild-type virus or by viruses with only "late" temperature-sensitive lesions (LA7, LA334R) is not significantly affected at elevated temperature. Reduction of viral D N A synthesis should affect the synthesis of viral RNA. As expected, the concentration of viral R N A is reduced at least five- to ten-fold in cells infected at the nonpermissive temperature with LA335, LA336, LA337 and LA338, whereas under

22 TABLE VI BIOLOGICAL PROPERTIES AND IN VITRO DNA SYNTHESIS OF SEVERAL TEMPERATURE-SENSITIVE MUTANTS OF AVIAN SARCOMA VIRUSES AND THEIR WILD-TYPE PARENT For details, see ref. 38. Efficiency of focus formation = focus forming titer of the virus at 41°C over focus forming titer at 35°C. Efficiency of replication = the ratio of the titer of virus formation at 41°C to the titer of the virus produced at 35°C. Virus harvested 72 h post-infection. "Minus" DNA indicates results obtained with labeled 70 S RNA as a hybridization reagent. "Plus" DNA indicates results obtained with labeled cDNA as hybridization reagent. Biological properties Efficiency of --Efficiency of . focus formation replication at 41°C at 41°C Wild-type LA335 LA336 LA337 LA338

0.24 0.002 0.001 0.006 0.001

1.10 0.001 0.001 0.004 0.002

.

DNA synthesis at 41°C DI~A synthesis at 35°C . . . . . . . . "'minus" DNA

"plus" DNA

1.8 0.1 0.1 0.06 0.16

I .3 0 12 0.09 0.13 0.12

similar conditions, the concentration of viral R N A was unaffected in cells infected by wild-type virus or by viruses bearing only late lesions (Bishop, J. M., Friis, R. R., Hunter, E and Valmus, H. E., personal communication). An additional mutant o f RSV, L A 3 3 8 [22,136,137], with both early and late temperature-sensitive lesions is deficient in viral D N A synthesis upon infection of cells at the nonpermissive temperature. However, the D N A polymerase of mutant L A 3 3 8 does not exhibit enhanced thermolability in vitro, as c o m p a r e d to that o f the parental enzyme [38]. Thus, this m u t a n t may be affected in some step prior to transcription of the viral genome into c D N A . Table VI summarizes the available data on the biochemical and biological characterization of "early" temperature-sensitive mutants of avian sarcoma viruses. There is a quantitative discrepancy between the biological properties of mutants with "early" lesions and biochemical properties, as manifested by thermolabile reverse transcriptase and impaired viral D N A synthesis at the nonpermissive temperature. Since the biochemical experiments wele carried out at high multiplicity of infection there might be enhanced leakiness o f the mutant viruses. When control experiments to study the biological properties were carried out at high multiplicity o f infection in parallel to biochemical experiments, increased leakiness of the mutants was observed (Hunter, E., personal communication). However, the efficiency of infection at 41°C under these conditions was generally still less than 0.02. The residual D N A synthesis seen at 41 °C may therefore represent partial transcription o f the viral genome prior to complete heat inactivation o f the reverse transcriptase. Three temperature-sensitive mutants of the Rauscher strain of murine leukemia virus (tsl 7, tsl9 and ts29) are defective in early functions required both for leukemia

23 TABLE VII AVERAGE HALF-TIME (t~) OF DNA POLYMERASE AND RNAase H ACTIVITIES OF A TEMPERATURE-SENSITIVE MUTANT OF R-MuLV AND ITS WILD-TYPE PARENT (t~) is the time required for 50~ inactivation of enzyme activity at a given temperature. For details see ref. 135. (t~) in rain at 45°C

Wild-type R°MuLV ts29

RNA-directed DNA polymerase

DNA-directed DNA polymerase

Ribonuclease

6.0 3.6

10.0 4.2

16.0 5.0

H

virus infection and helper function for rescuing mouse sarcoma viruses at nonpermissive temperature (39 °C) [138-140]. These mutants do not synthesize viral antigens or viral particles [139] at the nonpermissive temperature. Reverse transcriptase from one of these mutants, ts29, has been shown to be more thermolabile than the reverse transcriptase from the corresponding wild-type parent [141]. Both the D N A polymerase and RNAase H activities show enhanced thermolability [135]. The covariation of thermolability of D N A polymerase and RNAase H activities of ts29 suggests that like avian R N A tumor viruses, the two activities are an integral part of the reverse transcriptase activity of murine leukemia viruses. Two other early mutants rsl7 and tsl9, however, do not show more thermolabile reverse transcriptase than the wild-type enzyme. It should be interesting to study whether the cells infected with tsl7, tsl9 or is29, are also deficient in viral D N A synthesis at nonpermissive temperature. The average time of inactivation of 50 ~ of enzyme activity associated with purified reverse transcriptase from ts29 and parental wild type are shown in Table VII.

IX. THE CASE OF LA672

LA672 is a replication defective temperature-sensitive mutant of RSV [142,143] which produces large amounts of noninfectious viral particles during growth in cells at the nonpermissive temperature. This mutant may also have a temperaturesensitive lesion in the virion D N A polymerase, but with a physiology completely differentfromthatofLA335,LA336andLA337. Progeny synthesis can be manipulated at any time after infection by appropriate temperature shifts. The noninfectious virions of LA672 produced at 41 °C lack demonstrable reverse transcliptase activity. However, the reverse transcriptase of LA672 synthesized at 35 °C is not temperaturesensitive, nor are the virions produced under permissive conditions any more thermolabile than the wild-type virus [142]. The absence of polymerase activity from virus produced at 41 °C may therefore be due to a defect in production or packaging of the enzyme. LA672 can be complemented by avian leukosis viruses at 41°C, but if cells

24 infected with the known temperature-sensitive reverse transcriptase mutants, LA335 and LA337, are cocultivated with LA672 infected cells at 35 °C for 48 h and then shifted to 41°C, the progeny virus show a greatly reduced infectivity at 41 °C [142]. This observation suggests that the temperature-sensitive lesion in LA672 may affect the polymerase molecule directly, rather than interfel e with the packaging of an otherwise functional enzyme. However, it has recently been reported that there is enzyme in LA672 progeny made at the nonpermissive temperature because it is able to interfere with the neutralization of a wild-type enzyme with anti-reverse transcriptase antibodies (M6elling, K., personal communication). Characterization of the lesion of LA672 should prove useful in elucidating the steps in the biogenesis of the reverse transcriptase.

X, RSVp(-) In preparations of envelope-defective mutants of avian sarcoma viruses, an additional defect in the reverse transcriptase can be found. These are referred to as RSVp(--) (present in stocks of envelope-defective RSV(--)) and NY8~ (present in envelope-defective stocks of NY8) [22,144]. The defect in the reverse transcriptase cannot be complemented in cells which show partial expression of endogenous leukosis virus and which are positive for internal group specific virion antigens and for envelope glycoprotein [145]. The defect in RSVp(--) and NYS~ can be complemented by helper leukosis virus [145]. Characterization of RSVp(--) particles revealed that these are deficient in reverse transcriptase activity [77,99,144]. Furthermore, no immunological cross-reaction was observed when reacted with monospecific antibodies made against partially purified AMV D N A polymerase [77,99]. Thus it appears that RSVp(--) has a defect in the reverse transcriptase gene. In accord with the possibility that the defect is a deletion in reverse transcriptase gene is the fact that RSVp(--) shows no spontaneous reversion to wild type [22,145]. Biochemical evidence, however, suggests that this putative deletion is rather small as the RNA from RSVp(--) is not significantly smaller than the wild-type RNA [22]. Robinson and Robinson [146] have reported that a variant of Bryan high titer strain of RSV lacks RNA-dependent DNA polymerase activity but has intact DNAdependent D N A polymerase activity in the noninfectious particles. Since the two activities associated with purified DNA polymerase of virions have not yet been separated, it is not clear whether the DNA-dependent DNA polymerase activity represents viral or cellular DNA polymerase.

XI. MOUSE SARCOMA VIRUSES Mouse sarcoma viruses are defective and require helper leukemia viruses for replication [147,148]. The genome of MSV appears to be 4 to 6 kb as compared to

25 that of 9 to 10 kb of the helper virus genome [149-152]. Although there is no formal proof, it appears that MSV genome is unable to code for envelope glycoprotein and reverse transcriptase [22,95,96,153,154]. Several arguments favor this notion. (i) Noninfectious MSV is either quantitatively deficient or totally deficient in reverse transcriptase [155]. The small amounts of reverse transcriptase activity associated with particles released from sarcoma positive and leukemia negative cells may be due to either residual helper virus or endogenous virus. (iJ) A temperature-sensitive mutant of R-MuLV, ts29, with a defect in reverse transcriptase activity [141] is unable to rescue MSV from infected cells at nonpermissive temperature [140]. (iii) Reverse transcriptase isolated from MSV rescued by superinfection with RD-114, cross-reacts with antibodies raised against RD-114 reverse transcriptase. It had little or no cross-reaction with antibodies against murine leukemia virus reverse transcriptase [95]. (iv) If it is assumed that the gene order for avian sarcoma virus (gag-pol-env-src) [22] is the same for murine RNA tumor viruses, then heteroduplexes formed between MSV RNA and full length MLV cDNA show deletion loops of cDNA in the regions corresponding to polymerase and perhaps envelope regions [152]. A noninfectious hamster C-type RNA tumor virus (D9) associated with spontaneous hamster lymphomas has also been reported to lack reverse transcriptase activity [156]. XII. INTRACELLULAR REVERSE TRANSCRIPTASES Reverse transcriptases isolated from virions of RNA tumor viruses have been characterized extensively. Little progress has been made in the isolation and characterization of reverse transcriptase from cells infected with RNA tumor viruses. It is, of course, a more tedious chore to isolate reverse transcriptase from cells than it is from virions. Attempts to purify reverse transcriptase from cells are complicated by the presence of cellular D N A polymerases, nucleases and proteases. Generally, three types of cells have been examined: (a) ceils which were infected by a leukovirus and producing virus; (b) virus-infected cells which are nonproducers; and (c) cells which had no evidence for the presence of a virus. Two different biochemical approaches have been employed. The most common approach has been to look at the soluble enzyme activities by means of template requirements and physical and antigenic properties to show that the D N A polymerase is related to the virion reverse transcriptase. Anothel approach has been to look for a sedimentable RNA-directed D N A polymerase activity. In this case, the relatedness to the virion reverse transcriptase was checked for the nature of template, relationship to host or viral D N A polymerase, and the nature of the nucleic acid in the particle.

XIIA. Reverse transcriptase in infected cells producing virus Intracellular reverse transcriptases have been isolated from various cells

26 producing RNA tumor viruses, such as RSV-infected cells [157], BALB/3T3 cells producing M-MSV (MuLV) [158], mouse bone marrow cells infected with M-MuLV [159], mouse spleen cells infected by R-MuLV [160], NC37 cells infected by and producing simian sarcoma virus-I or GaLV [161], IdU-induced BALB/c 3T3 ceils and mammalian cells (human, dog and mink) chronically producing RD-114 virus [162]. The enzymes are isolated either from cell homogenates oi from the microsomal pellet fraction. Coffin and Temin [157] have shown that a cytoplasmic particulate fraction isolated from detergent disrupted virus producing chicken cells contained a DNA polymerase activity. In contrast to the virions, the DNA synthesis in the particulate was not sensitive to RNAase treatment. However, the product DNA was probably synthesized via an RNA template, since the DNA ploduct could anneal to the RNA isolated from purified virions. Ross et al. [158] purified DNA polymerase from BALB/3T3 cells infected with MSV (MuLV) by a combination of gel filtration and ion exchange chromatography. Three peaks of enzymatic activity were found in virus-infected cells as compared to the two from uninfected cells. The additional enzyme activity from virus-infected cells behaved Jn chromatographic properties and template preferences like enzyme isolated from purified murine leukemia virus. Similarly, Batlimore et al. [159] have shown that cells infected with and producing murine leukemia virus have an obvious DNA polymerase activity which chromatographs differently from any of the cellular DNA polymerases and is easily detected by its ability to utilize poly(C).oligo(dG) as template-primer. No such enzymatic activity could be found in the extracts of uninfected cells. Gerwin et al. [162] used a novel approach to isolate reverse transcrJptase from infected cells by affinity columns. They purified reverse transcriptase from RD-114 infected human amnion cells by affinity chromatography on oligo(dT) cellulose columns. The molecular weight of intracellular reverse transcriptase of RD-114 virus was 95 000. This is Jn contrast to the molecular weight of 70 000 attributed to purified reverse transcriptase fiom RD-114 virus. It was speculated that the larger form replesents a cellular precursor to the smaller species which is packaged into the RD114 virion. It should be pointed out that none of these studies have unambiguously ruled out contamination by membrane-bound virus particles or immature virions. XIIB. Reverse transcriptase in nonprodueer-infected cells The isolation of an RNAase-sensitive DNA polymerase activity from rat cells infected by RSV has been reported [163]. These cells were transformed, and contained the complete viral genome [164]. However, these cells did not contain or produce infectious virus. The polymerase activity required all four deoxyribonucleoside triphosphates. Surprisingly, the product DNA from this polymerase activity did not hybridize with RNA isolated from purified virions of the B77 virus used for the original infection of the rat cells. Furthermore, it did not hybridize with the RNA of

27 an endogenous C-type RNA virus of rat cells, R-35 virus, or murine sarcoma virus. The product DNA was shown to hybridize to some extent to RNA from either uninfected or infected rat t,ells. Antibodies raised against AMV DNA polymerase did not inhibit the activity of this DNA polymerase [5]. It is not clear if this reverse transcriptase-like activity is cellular or viral in origin. Livingston et al. [165] reported the presence of a new DNA polymerase activity, termed peak A, from the cytoplasmic pellet fraction of nonproducer Kirsten-MSV infected BALB/3T3 cells, termed KA31. Peak A eluted from a phosphocellulose colum at 0.22 M KC1 and resembled viral reverse transcriptase in chromatographic behavior, molecular weight of 70 000, ability to utilize poly(A).oligo(dT) as templateprimer, and partial neutralization with anti-R-MuLV antibodies. However, peak A was unable to transcribe either 70 S viral RNA or poly(C).oligo(dG). The biological significance of peak A remains to be investigated as is its detailed biochemical characterization. XIIC. Reverse transcriptase in normal cells Several reports have described the presence of RNA-dependent DNA polymerase activity in normal or uninfected cells. However, none of these reports has sufficiently characterized the enzyme from normal cells to allow comparisons to be made with authentic viral reverse transcriptases. Coffin and Temin [163] have reported the presence of a sedimentable ribonuclease sensitive DNA polymerase activity from normal rat embryo fibroblasts in culture. A sedimentable endogenous RNA-directed DNA polymerase activity has also been reported in normal chicken embryo cells [166]. This activity was RNAase-sensitive, but was not affected by DNAase. DNA.RNA hybrids are formed in a short-term endogenous reaction and the DNA product can hybridize to RNA isolated from this fraction. The product DNA does not hybridize to ALSV RNA or REV RNA. The endogenous RNAdirected DNA polymerase activity is partially or completely neutralized by antibodies raised against small DNA polymerase isolated from chicken embryos [166a] but was not affected by anti-AMV reverse transcriptase antibodies. It will be important to determine whether this activity can transcribe 70 S viral RNA or utilize poly(C)-oligo(dG) as template-primer and whether it has an RNAase H activity associated with it. Mayer et al. [167] have reported the isolation and characterization of reverse transcriptase-like activity from the normal rhesus monkey placenta. The enzyme was antigenically closely related to reverse transcriptase from M-7 virus, an isolate obtained through cocultivation of baboon placental cells with heterologous cells [168]. It has also been reported that Type C virus can be seen in placentas and fetal tissues of rhesus monkeys [169]. Thus it is possible that the results of Mayer et al. [167] are due to the presence of reverse t:anscriptase activity of an endogenous primate virus. Another report of the presence of RNA-directed DNA polymerase activity in normal cells comes from the studies of ribosomal DNA (rDNA) in Xenopus laevis orcytes. During the early stages of orgenesis in X. laevis, the DNA which codes for

28 28 S and 18 S ribosomal RNA has been shown to undergo a 1000-fold amplification [170-172]. It has been demonstrated that gene amplification proceeds through a chromosome copy mechanism [173]. One possible mechanism to account for rDNA gene amplification was proposed by Crippa and Tocchini-Valentini [174]. This involves the transcription of the complete RNA copy of the rDNA monomei by an RNA-dependent DNA polymerase. They have identified nascent DNA chains associated with 45 S rRNA precursor [175]. The synthesis of DNA was RNAase sensitive and the DNA dissociated upon heating The DNA product appears to have some RNA covalently attached to it (Tocchini-Valentini, G. P., personal communication). Brown and Tocchini-Valentini [175,176] have isolated RNA-directed DNA polymerase from the ovaries of X. laevis. The ovaries were homogenized and centrifuged to obtain 100 000 g supernatant. The proteins were precipitated with ammonium sulfate, followed by sequential chromatography on DEAE-Sephadex, phosphocellulose and DNA cellulose. The resulting enzyme has an approximate molecular weight of 100 000 and can utilize l0 S globin mRNA as template to synthesize complementary DNA. The product of cDNA is very small but heteropolymeric regions are transcribed because the reaction requiles the presence of all four deoxyribonucleoside triphosphates. Furthermore, synthesis of cDNA to globin mRNA is actinomycin D-sensitive. This enzyme appears to have different ion requirements and template specificities than the cellular DNA polymerases isolated from amphibian o6cytes. It is unlikely, but the possibility that amphibian o6cytes used to isolate the enzyme may have C-type particles has not been ruled out (Tocchini-Valentini, G. P., personal communication). Bird et al. [177] have reported their inability to find 45 S RNA in o6cytes involved in the synthesis of rDNA. Moreover, they conclude that the [3H]uridine could be incorporated into DNA after conversion to deoxycytidine by the ovaries. This, however, does not appear to be the likely reason for the discrepancy between the results of the two groups as the synthesis of DNA associated with 45 S RNA is RNAase sensitive. Tocchini-Valentini et al. [175] have suggested that the discrepancy could be due to the use of frogs by Bird et al. [175] which are at a later stage of the amplification process. Furthermore, Tocchini-Valentini et al. were able to show that indeed, ovaries of tadpoles from later stages of the rDNA amplification process, did not exhibit RNAase-sensitive newly synthesized rDNA. The observations of Tocchini-Valentini's group are very provocative, but more work will be required to prove the unambiguous presence of reverse transcriptase-like activity in amphibian o6cytes. XIID. Reverse transcriptase in tumor cells

Gallo and his associates have isolated reverse transcriptase-like activity from human leukemic cells [11,12,178]. The cells were obtained from patients with acute myelogenous leukemia and the enzyme purified from cytoplasmic particulate fractions. Partially purified enzyme exhibits biochemical and immunological properties similar to those of the known murine and primate virus reverse transcriptases [102,161,179].

29 The purified enzyme [180] is capable of utilizing poly(C).oligo(dG) as template RNA. It can also transcribe heteropolymeric portions of both AMV and mammalian 60-70 S viral RNA. The molecular weight of reverse transcriptase from human leukemic cells was found to be 130 000 in low salt and 70 000 in high salt. The two forms of enzyme are interconvertible and appear to manifest different efficiencies of transcription of heteropolymers [180]. It will be of interest to find out if this enzyme has also an associated RNAase H activity. Crude extracts made from skin of Xeroderma pigmentosum patients were shown to stimulate the incorporation of labeled dGTP with poly(dC).poly(G) as template [181]. Similar extracts from the skin of normal people were unable to utilize this template. However, the choice of poly(dC) as template does not rule out that the observed activity is due to DNA-dependent DNA polymerase rather than RNAdependent DNA polymerase. The presence of reverse transcriptase-like activity in human milk particles [182], human leukemic cells and other malignant cells [183-185) has been shown by simultaneous detection techniques. This technique involves preparing a high speed pellet and then treating the pellet with nonionic detergent and incubating the mixture with substrates. The DNA product of the reaction is analyzed to see if it sediments at a 60-70 S complex and bands at a density characteristic of an RNA.DNA hybrid. No reverse transcriptase-like activity has been isolated or characterized from such systems. Results obtained by using the simultaneous detection technique, however, are often difficult to interpret unambiguously. The possible problems have been discussed extensively by Sarngadharan et al. [12].

xIII. DIAGNOSTIC FEATURES OF REVERSE TRANSCRIPTASE It was generally assumed that the ability to transcribe ribopolymers is characteristic of reverse transcriptase activity. However, subsequent studies with cellular DNA polymerases showed that at least one species of cellular DNA polymerases, DNA polymerase 7, can also utilize ribopolymer, poly(A), to synthesize complementary poly(dT) [11,12,186]. Furthermore, reverse transcfiptase from mammalian viruses appears to transcribe poly(dC).oligo(dG) more efficiently than poly(C).oligo(dG) [41]. I do not believe that there is an absolute test for reverse transcriptase activity. However, enough information is available to make general remarks about the criteria for defining reveise transcriptase activity. They are as follows: (a) ability to transcribe ribohomopolymers and riboheteropolymers. (b) Ability to utilize poly(C).oligo(dG) as template primer. Recently Gerard et al. [187] have shown that poly(2'-O-methylcytidylate).oligo(dG) is a very efficient template.primer for reverse transcriptase from avian, murine, feline and primate RNA tumor viruses. None of the cellular DNA polymerases from either prokaryotic or eukaryotic origin was able to transcribe it. (c) In general, reverse transcriptase should transcribe ribopolymers more efficiently than deoxyribopolymels. Poly(dA).oligo(dT) is very

30 efficiently transcribed by cellular DNA polymerases but often is not even transcribed by reverse transcriptases. (d) Association of RNAase H activity with reverse transcriptase. Viral reverse transcriptase associated RNAase H activity is an exonuclease. (e) Cross-reactivity with antiserum raised against some known viral reverse transcriptase.

XIV. REVERSE TRANSCRIPTION XIVA. Viral R N A as template

When purified virions are lyzed with nonionic detergent and incubated with substrate, DNA complementary to viral RNA is synthesized. The average size of the cDNA synthesized is generally between 200 and 400 nucleotide long [5], as compared to the size cf the viral genome of 7.5 to 10 kb. Although the size of the cDNA synthesized is very short, all portions of the RNA genome are transcribed [18]. Since the eDNA synthesized was representative of the whole genome, its small size was largely ignored as an annoyance. Recently, however, several laboratories have reported the synthesis of long transcripts of eDNA approaching the length of the viral genome [188-191]. It is not clear whether the ability to synthesize full genome length cDNA transcripts is due to high substrate concentrations (1-5 mM deoxynucleoside triphosphates) employed in the reaction or optimal conditions for the lysis of the viruses. We have found that the yield of the full length cDNA transcript is maximum if freshly harvested viruses are used. Generally, the amount of the full length cDNA transcripts represent only 2-5 ~o of the total eDNA synthesized. Fig. 6A shows the sedimentation analysis of the cDNA synthesized from purified virions of AMV. Fig. 6B shows the sedimentation of fraction I isolated from total eDNA in Fig. 6A. It appears to represent full genome length eDNA transcripts of average size larger than 6.2 kb. The genome of AMV is approximately 7.5 kb long. Similarly, full genome length cDNA transcripts have been obtained from MuLV and RSV (Verma, I. M. and Vogt, P. K., unpublished results). The mechanism of initiation of DNA synthesis and forms of the cDNA synthesized are the subjects of accompanying articles. XIVB. Natural R N A s as templates

Purified reverse transcriptase can faithfully transcribe polyribonucleotides into polydeoxyribonucleotides if an appropriate primer is supplied to initiate DNA synthesis. This versatility of the enzyme has proven to be a boon to molecular biologists. Many eukaryotic mRNAs have been transcribed into complementary DNA [25,192-199]. Advantage has been taken of the fact that most eukaryotic mRNAs contain as an integral part of their structure poly(A) tails at their 3'-OH end. An oligomer of (dT) can hydrogen bond to the poly(A) and serve as primer to initiate the synthesis of cDNA. Generally, the cDNA is synthesized in the presence of acinomycin D, a drug which prevents the synthesis of the second strand of DNA.

31 ~._~~ 18S 16S

7S

,ooo., 11 2 0 0 0 ~"

1500 l.iJ I.--

IOOO

500 IZ Z) 0 o "1-

~_~

0

,

.

.

B

.

.

.

.

.

.

.

IM I---

I8S 16S

~

400

Z

~

%

I

ioo .~ ir i~J a.

,q--- M A R K E R

09 iz

2oo

[~'i

~RAC~,O.

50 o( D

?

o! 0

i

~'

.

~-.~

IO 20 50 FRACTION N U M B E R

~40

Fig. 6. Sedimentation profile of cDNA synthesized to AMV RNA on alkaline sucrose gradients (A) Total eDNA synthesized: The reaction mixture contained 50 mM Tris. HCI, pH 8.3, 10 mM dithiothoeitol, 6 mM magnesium acetate, 60 mM NaC1, 2 mM each of dATP, dCTP, dGTP and 3H dideoxythymidine triphosphate. 3H- labeled dideoxythymidine triphosphate was used at a specific activity of 100 Epm per pmol to monitor the progress of the reaction. 200/zg of purified virions were used. Nonidet P-40 at a final concentration of 0.2-0.3 ~ was used to lyse the virions. The reaction mixture was incubated for 360 rain at 37°C and fractionated on a G-75 Sephadex column. Fractions appearing in the void volume were combined and nucleic acids extracted with phenol/chloroform, followed by precipitation with 2 volumes of ethanol. The sample was dissolved in 0.6 ml of alkaline buffer containing 0.3 M KOH, 0.7 M LiC1, 0.005 M EDTA, pH 12.5, and layered on a 10.8 ml 15-30 ~ sucrose gradient made in the alkaline buffer. Either in the sample gradient or in a parallel gradient, form II of polyoma virus DNA (kindly given by Dr. M. ¥ogt) was added as a standard marker. The samples were centrifuged in rotor SW41 at 32 000 rev./min for 15 h at 20°C. About 40--45 fractions of 0.27 ml each were collected by puncturing the bottom of the gradient with a needle. The samples were neutralized and radioactivity counted in 3.0 ml of aquasol. The standard sediments at 17.8 S (form II circles) and 16 S (unit length). (B) Resedimentation of fraction 1. Fractions marked under the area 1 (fraction 1) were pooled, neutralized and precipitated with 2 volumes of ethanol. The sample was dissolved in alkaline buffer and centrifuged as above.

Since e D N A copies have been extensively utilized in hybridization techniques, it is useful to have single-stranded e D N A copies. If, however, aetinomycin D is omitted from the reaction mixture containing, for instance, globin m R N A as template, doublestranded D N A copies are synthesized [195]. It appears that single-stranded e D N A folds back on itself and can provide a 3'-OH end for the synthesis o f the second strand

32 [195]. Recently double-stranded cDNA to globin mRNA has been synthesized by using single-stranded cDNA copies as template, and E. coli DNA polymerase 1 to make the second strand [200]. The size of the cDNA synthesized depends upon the nature of the template and the substrate concentration. It has been shown that in the presence of high deoxyribonucleoside trophosphates, full length cDNA copies can be synthesized [201]. However, it has been very difficult to synthesize full length cDNA copies in 14 S immunoglobulin mRNA (Baltimore, D., personal communication, and Stavnezer, J. and Bishop, J. M., personal communication). Perhaps the secondary structure of the mRNA limits its availability as a template for transcription. In a recent publication, Kacian and Meyers [199] have shown that nearly full length cDNA to 35 S polio RNA can be synthesized. The requirement of high substrate concentration can be overcome by 1 mM each of ribonucleoside triphosphates or by adding 0.4 mM sodium pyrophosphate to the reaction mixture. Drost, S. and Baltimore, D. (personal communication) have shown also that the presence of NaF allows the synthesis of full length cDNA to polio RNA. It appears that purified preparations of reverse transcriptase contain some nuclease or phosphatase activity which can be reduced by the addition of ribonucleoside triphosphates, NaF or sodium pyrophosphate. It thus appears that in the future synthesis of full template length cDNA transcripts should be the norm rather than being an exception.

X1VC. Transcription of RNAs lacking poly(A). Reverse transcriptase will transcribe any given RNA template, as long as a proper primer is provided to initiate DNA synthesis. Many RNAs have C-rich or G-rich regions which can base pair with an oligomer of complementary oligonucleotide to provide a 3'-OH end to initiate transcription. We have found that 28 S and 18 S mRNA from HeLa cells (Verma, I. M. and Weinberg, R. A., unpublished results) or from cellular slime molds (Verma, I. M. and Firtel, R. A., unpublished results) could be reverse transcribed by using an oligomer of dC to prime the synthesis o f c D N A . However, the size of the product cDNA will depend upon the location of the C- rich or G-rich regions on the RNA. As mentioned earlier, deoxyribo oligomers are more efficient primers than ribo oligomers [204]. Recently it has been shown that DNAase digested calf thymus DNA (tetranucleotide long) can act as primer to initiate D N A synthesis [202]. For those RNA species which lack poly(A) or A, C, G, or U-rich regions, perhaps DNAase digested calf thymus DNA can be used as primer. Another approach will be to add a stretch of poly(A) or any other nucleotide as the Y-OH end of a given RNA [203].

XV. FUTURE RESEARCH TRENDS ON REVERSE TRANSCRIPTASE In the half a dozen years since the discovery of reverse transcriptase, we have learned a great deal about its properties, purification, structure and role in the life

33 cycle of RNA tumor viruses. In the next half a dozen years, I think we will witness more detailed and fine analysis of its synthesis in infected cells, structure, mechanism of action in vivo and in vitro. Because of the availability of large quantities of reverse transcriptase from avian RNA tumor viruses, this enzyme will probably be used extensively for most structural studies. Many laboratories will try to isolate the native fir or fl forms of the enzyme and study their in vitro conversion to the aft and a forms of the enzyme. We would like to ask the following kinds of questions: does a originate from a cleavage of the N-terminal or C-terminal or both ends of r? Is the intracellular enzyme in fir dimer form? Is the enzyme packaged in the virion as fir dimer or as aft (the most abundant and stable form)? Does the enzyme itself contain a proteolytic activity to allow the conversion of fir to aft form? It appears that RNAase H activity is an integral part of reverse transcriptase isolated from avian, murine or hamster RNA tumor viruses. I think a major effort will be made by many investigators to study what role the associated RNAase H activity plays in the synthesis of the provirus. We would like to know if all reverse transcriptases contain RNAase H activity. It its always an exoribonuclease? Can RNAase H activity be physically separated from DNA polymerase activity? Attempts will be made to isolate temperature-sensitive mutants of RNA tumor viruses with a more pronounced lesion in RNAase H activity than the DNA polymerase activity. The mechanism by which reverse transcriptase synthesizes a double-stranded circular proviral DNA is alrgely unknown. How does the synthesis of eDNA initiated near the 5'-end of the genome shift to synthesis of cDNA from the 3'-OH end of the genome? When does the synthesis of second strand of DNA begin? Does the synthesis of the second strand of DNA also require a primer? What is the nature of the template RNA during or after the synthesis of proviral DNA? Finally, I think we will also begin to learn about the synthesis of reverse transcriptase following infection. The availability of monospecific antibodies to reverse transcriptase and the development of radioimmunoassays should prove very useful for these studies. One would like to know if all reverse transcriptases are present in the infected cell as dimer forms of their subunits. In other words, are all reverse transcriptases two-subunit complexes? In what form is the mature reverse transcriptase generally packaged in the virion? It should be interesting to see if the defect in LA672 is in the packaging or processing of reverse transcriptase.

ACKNOWLEDGEMENTS I am very grateful to David Baltimore, Walter Eckhart, Hung Fan, Bertold Francke, M. T. Lai, Satoshi Mizutani, Bart Sefton, Howard Temin and Robert Wells for numerous suggestions and instructive criticism of this manuscript. It is always a great pleasure to acknowledge the members of the Tumol ' Virology Labora-

34 tory for stimulating discussions.

I a m deeply indebted to C a r o l y n G o l l e r f o r the

invaluable help in the p r e p a r a t i o n o f this manuscript.

This w o r k was s u p p o r t e d by

Public H e a l t h Service G r a n t N o . C A 16561-01.

REFERENCES 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Baltimore, D. (1970) Nature 226, 1209-1211 Temin, H. and Mizutani, S. (1970) Nature 226, 1211-1213 Temin, H. M. (1974) Natl. Cancer Inst. Monogr. 17, 557 Bader, J. P. (1965) Virology 26, 253 Temin, H. M. and Baltimore, D. (1972) in Advances in Virus Research (Smith, K, M. and Lauffer, M. A., eds.), Vol. 17, pp. 129-186, Academic Press, New York Temin, H. M. (1974) Annu. Rev. Gen. 8, 155-177 Temin, H. M. (1972) Sci. Am. 226, 24--33 Tooze, J. (ed.) (1973) Molecular Biology of Tumor Viruses, Chapts. 10, 11, Cold Spring Harbor Laboratory, New York Sarin, P. S. and Gallo, R. C. (1974) in Nucleic Acids, International Review of Science series in Biochemistry (Burton, K., ed.), Vol. 6, p. 219, Butterworth and Medical and Technical Publishing Co., Oxford Green, M. and Gerard, G. F. (1974) Progr. Nucl. Acid Res. Mol. Biol. 14, 187-334 Wu, A. M. and Gallo, R. C. (1975) CRC Critical Rev. Biochem. pp. 289-347 Sarngadharan, M. G., Allaudeen, H. S. and Ga.lo, R. C. (1976) Methods Cancer Res. 12, 3-47 Varmus, H. E., Guntaka, R. V., Deng, C. T. and Bishop, J. M. (1975) Cold Spring Harbor Symp. Quant. Biol, 39, 987-996 Gianni, A. M., Smotkin, D. and Weinberg, R. A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 447--451 Jacquet, M., Groner, Y., Monroy, G. and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3045-3049 Rymo, L., Parsons, J. T., Coffin, J. M. and Weissmann, C. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2782-2786 Astrin, S. M. (1974) Biochem. Biophys. Res. Commun. 61, 1242-1248 Fan, H. and Baltimore, D. (1973) J. Mol. Biol. 83, 93-117 Gielkens, A. L. J., Salden, M. H. L. and Bloemendal, H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1903-1097 Schincariol, A. L. and Joklik, W. K. (1973) Virology 56, 532 Bishop, J. M., et al (1976) ICN-UCLA Syrup. Mol. Cell. Biol., Animal Virol. (Baltimore, D., Huang, A. S. and Fox, C. F., eds.), in the press Vogt, P. K. (1976) The Genetics of RNA Tumor Viruses in Comprehensive Virology (FraenkelConrat, H. and Wagner, R. R., eds.), in the press Nowinski, R. C., Sarkar, N. H. and Fleissner, E. (1973) in Methods in Cancer Research (Busch, H. ed.), Vol. 8, p. 237, Academic Press, New York Kacian, D. L., Watson, K. F., Burny, A. and Spiegelman, S. (1971) Biochim. Biophys. Acta 246, 365-383 Verma, I. M. and Baltimore, D. (1974) in Methods in Enzymology (Grossman, L. and Moldave, K., eds.), Vol. 29, pp. 125-131: Academic Press, New York Grandgenett, D. P., Gerard, G. F. and Green, M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 230--234 Faras, A. J., Taylor, J. M., McDonnell, J. P., Levinson, W. E. and Bishop, J. M. (1972) Biochemistry 11, 2334-2342 Hurwitz, J. and Leis, J. P. (1972) .1. Virol. 9, 116-129 Wang, L. H. and Duesberg, P. H. (1973) J. Virol. 12, 1512-1521 Abrell, J. W. and GaUo, R. C. (1973) J. Virol. 12, 431 Verma, I. M., Meuth, N. L., Fan, H. and Baltimore, D. (1974) J. Virol. 13, 1075-1082 Verma, I. M. (1975) J. Virol. 15, 843-854 Panet, A., Verma, I. M. and Baltimore, D. (1975) Role of the Subunits of the Avian RNA Tumor Virus Reverse Transcriptase, Cold Spring Harbor Syrup. Quant. Biol. 39, 919-923 Gerwin, B. I. and Milstein, J. B. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2599-2603

35 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

Grandgenett, D. P. and Rho, H. M. (1975) J. Virol. 15, 526-533 Grandgenett, D. P., J. Virol., in the press Marcus, S. L., Modak, M. J. and Cavalieri, L. F. (1974) J. Virol. 14, 853-859 Verma, I. M., Varmus, H. E. and Hunter, E. (1976) Virology, in the press Chirikjian, J. G., Rye, L. and Papas, T. S. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1142 Goodman, N. C. and Spiegelman, S. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2203 Baltimore, D. and Smoler, D. F. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1507-1511 Verma, I. M., Mason, W. S., Drost, S. D. and Baltimore, D. (1974) Nature 251, 27-31 Verma, I. M., Meuth, N. L., Bromfeld, E., Manly, K. and Baltimore, D. (1971) Nat. New Biol. 233, 131-134 Faras, A. J., Dahlberg, J. E., Sawyer, R. C., Harada, F., Taylor, J. M., Levinson, W. E., Bishop, J. M. and Goodman, H. M. (1974) J. Virol. 13, 1134 Dahlberg, J. E., Harada, F. and Sawyer, R. C. (1975) Cold Spring Harbor Symp. Quant. Biol. 39, 925-932 Waters, L. C. (1975) Biochem. Biophys. Res. Commun. 65, 1130 Smoler, D., Molineaux, I. and Baltimore, D. (1971) J. Biol. Chem. 246, 7697 Springgate, C. F. and Loeb, L. A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 245 Battula, N. and Loeb, L. A. (1974) J. Biol. Chem. 249, 4086 Battula, N. and Loeb, L. A. (1976) J. Biol. Chem. 251,982 Brutlag, D. and Kornberg, A. (1972) J. Biol. Chem. 246, 241-248 Kornberg, A. (1975) D N A Synthesis, W. H. Freeman and Co., San Francisco Fansler, B. S. and Loeb, L. A. (1974) Methods in Enzymol. 24, 53-70 M611ing, K., Bolognesi, D. P., Batter, W., Biisen, W., Plassmann, H. W. and Hausen, P. (1971) Nat. New Biol. 234, 240-243 Baltimore, D. and Smoler, D. (1972) J. Biol. Chem. 247, 7282-7287 Leis, J., Berkower, I. and Hurwitz, J. (1973) in D N A Synthesis in vitro (Wells, R. D. and Inman, R. B., eds.), pp. 287-308, University Park Press, Baltimore Keller, W. and Crouch, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3360-3364 M611ing, K. (1976) J. Virol. 18, 418-425 Grandgenett, D. P. and Green, M. (1974) J. Biol. Chem. 249, 5148-5152 Brewer, L. and Wells, R. D. (1974) J. Virol. 14, 1494 Collett, M. S. and Faras, A. J. (1976) J. Virol. 17, 291 Mizutani, S., Boettiger, D. and Temin, H. M. (1970) Nature 228, 424 Duesberg, P., Helm, K. V. D. and Canaani, E. (1971) Proc. Natl. Acad. Sci. U.S. 68, 747 Strand, M. and August, J. T. (1971) Nat. New Biol. 233, 137 Mizutani, S. and Temin, H. M. (1971) J. Virol. 8, 409 Hung, P. P. (1971) Virology 51,287 Mizutani, S., Temin, H. M., Kodama, M. and Wells, R. D. (1971) Nat. New Biol. 230, 232 Miller, L. K. and Wells, R. D. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2298 Jovin, T. M., Englund, P. T. and Bertsch, L. L. (1969) J. Biol. Chem. 244, 2996-3008 Martin, R. G. and Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379 Verma, I. M. (1975) J. Virol. 15, 121-126 Gibson, W. and Verma, I. M. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4991-4994 M611ing, K. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 969-973 Papas, T. S., Marciani, D. J., Samuel, K. and Chirikjian, J. G. (1976) J. Virol. 18, 904--910 Nowinski, R. C., Watson, K. F., Yaniv, A. and Spiegelman, S. (1972) J. Virol. 10, 959-964 Rho, H., Grandgenett, D. P. and Green, M. (1975) J. Biol. Chem. 250, 5278-5280 Panet, A., Baltimore, D, and Hanafusa, T. (1975) J. Virol. 16, 146-152 Grandgenett, D. P. (1976) J. Virol. 17, 950 Grandgenett, D. P. (1976) ICN-UCLA Symp. Mol. Cell. Biol., Animal Virol. (Baltimore, D. and Huang, A. S., eds.), pp. 215-226 David, G. and Reisfeld, R. A. (1974) Biochemistry 13, 1014-1021 Carter, J. R., Fox, F. C. and Kennedy, E. P. (1968) Proc. Natl. Acad. Sci. U.S. 60, 725 Panet, A., Haseltine, W. A., Baltimore, D., Peters, G., Harada, F. and Dahlberg, J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2535-2539 Grandgenett, D. P., Vohra, A. K. and Faras, A. J. (1977) Virology, in the press

36 84 Purchase, H. G., Ludford, G., Nazarian, K. and Cox, H. W. (1973) J. Natl. Cancer Inst. 51, 489-499 85 Cooper, G. M. and Temin, H. M. (1974) J. Virol. 14, 1132-1141 86 Kang, C. Y. and Temin, H. M. (1974) J. Virol. 14, 1179-1188 87 Kieras, R. M. and Faras, A. J. (1975) Virology 65, 514-523 88 Waite, R. F. M. and Allen, P. T. (1975) J. Virol. 16, 872-879 89 Kang, C. Y. (1975) J. Virol. 16, 880-886 90 Mtilling, K., Gelderblom, G., Pauli, G., Friis, R. R. and Bauer, H. (1975) Virology 65, 546-557 91 Mizutani, S. and Temin, H. M. (1974) J. Virol. 13, 1020-1029 92 Mizutani, S. and Temin, H. M. (1975) J. Virol. 16, 797-806 93 M~311ing, K. (1974) Virology 62, 46-59 94 Gerard, G. F. and Grandgenett, D. P. (1975) J. Virol. 15, 785-797 95 Peebles, P. T., Gerwin, B. I. and Scolnick, E. M. (1976) Virology 70, 313-323 96 Peebles, P. T., Haapala, D. K. and Gazdar, A. F. (1972) J. Virol. 9, 488-493 97 Aaronson, S. A., Parks, W. P., Scolnick, E. M. and Todaro, G. J. (1971) Proc. Natl. Acad. Sci. U.S. 68,920-924 98 Parks, W. P., Scolnick, E. M., Ross, J. Todaro, G. J. and Aaronson, S. A. (1972) J. Virol. 9, 110-115 99 Hanafusa, H., Baltimore, D., Smoler, D. F., Watson, K. V., Yaniv, A. and Spiegelman, S. (1972) Science 177, 1188-1191 100 Scolnick, E. M., Parks, W. P., Todaro, G. J. and Aaronson, S. A. (1972) Nature 235, 35-40 101 Scolnick, E. M., Parks, W. P. and Todaro, G. J. (1972) Science 177, 1119 102 Todaro, G. J. and Gallo, R. C. (1973) Nature 244, 206 103 Livingston, D. M. and Todaro, G. J. (1973) Virology 53, 142 104 Yaniv, A., Ohno, T., Kacian, D., Colcher, D., Witkin, S., Schlom, J. and Spiegelman, S. (1974) Virology 59, 335 105 Gallo, R. C., Smith R. G. and Gallagher, R. E. (1975) Eukaryotic DNA Polymerase Conference at the Asilomar Conference Center in Monterey, California 106 Ting, R. C., Yang, S. S. and Gallo, R. C. (1972) Nat. New Biol. 236, 163 107 Gurgo, C., Ray, R. K., Thiry, L. and Green, M. (1971) Nat. New Biol. 229, 111 108 Yang, S. S., Herrera, F. M., Smith, R. G., Reitz, M. S., Lancini, G., Ting, R. C. and Gallo, R. C. (1972) J. Natl. Cancer inst. 49, 7 109 Gurgo, C., Ray, R. and Green, M. (1972) J. Natl. Cancer Inst. 49, 61 110 Brockman, W. W., Carter, W. A., Li, H., Rensser, F. and Nichol, F. R. (1971) Nature 230, 249 111 Carter, W. A., Brockman, W. W, and Borden, E. C. (1971) Nat. New Biol. 232, 212 112 Papas, T. S., Sandhaus, L., Chirigos, M. A. and Furusawa, E. (1973) Biochem. Biophya. Res. Commun. 52, 88 113 Papas, T. S., Pry, T. W. and Chirigos, M. A. (1974) Proc. Natl. Acad. Sci. U.S. 71, 367 114 Gurgo, C., Grandgenett, D. P., Gerard, G. F. and Green, M. (1974) Biochemistry 13, 708 115 McDonnell, J. P., Garapin, A., Levinson, W. E., Quintrell, N., Fanshier, L. and Bishop, J. M. (1970) Nature 228, 433 116 Muller, W. E. G., Zahn, R. K. and Seidel, H. J. (1971) Nat. New Biol. 232, 143 117 Apple, M. A. and Haskell, C. M. (1971) Physiol. Chem. Phys. 3, 307 118 Chandra, P., Zunino, F., Gotz, A., Gericke, D., Thorbeck, T. and DiMarco, A. O. (1972) FEBS Lett. 21,264 119 Hirschman, S. Z. (1971) Trans. N. Y. Acad. Sci. Ser. 11, 33, 595 120 Muller, W. E. G., Yamazaki, Z., Forster, W., Zahn, K. and Seidel, H. J. (1972) Klin. Wochenschr. 50, 990 121 Kotler, M. and Becker, Y. (1970) Nat. New Biol. 234, 212 122 Chandra, P., Zunino, F., Gorz, A., Wacker, A., Gericke, D., DiMarco, A., Casazza, A. M. and Giuliani, F. (1972) FEBS Lett. 21, 154 123 Hirschman, S. Z. (1971) Science 173, 441 124 Fridlender, B. and Weissbach, B. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3116 125 Tuominen, F. W. and Kenney, F. T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2198 126 Chirigos, M. A., Pearson, J. W., Papas, T. S., Woods, W. A., Woods, Jr., H. B. and Spahn, G. (1973) Cancer Chem. Rep. 57, 305

37 127 Muller, W. E. G., Yamazaki, Z. and Zahn, R. K. (1972) Biochem. Biophys. Res. Commun. 46, 1667 128 Levinson, W. E., Faras, A. J., Woodson, B., Jackson, J. and Bishop, J. M. 0973) Proc. Natl. Acad. Sci. U.S. 70, 164 129 Tuominen, F. W. and Kenney, T. (1972) Biochem. Biophys. Res. Commun. 48, 1469 130 Poiesz, B. J., Battula, N. and Loeb, L. A. (1974) Biochem. Biophys. Res. Commun. 56, 959 131 Auld, D. S., Kawaguchi, M , Livingston, D. M. and Vallee, B. L. (1975) Biochem. Biophys. Res. Commun. 62, 296 132 Linial, M. and Mason, W. S. (1973) Virology 53, 258-273 133 Mason, W. S., Friis, R. R., Linial, M. and Vogt, P. K. (1974) Virology 61, 559-574 134 Verma, I. M. and Gibson, W. (1975) in ICN-UCLA Symp. Mol. Cell. Biol., in D N A Synthesis and its Regulation (Goulian, M., Hanawalt, P. and Fox, C. F. (eds.), Vol. 3, pp. 730-752, W. A. Benjamin, Menlo Park, California 135 Verma, I. M. and Hunter, E. (1975) in Comparative Biochemistry, Molecular Biology and Immunology of R N A Tumor Viruses in Comparative Leukemia Research (Clemmesen, J. and Yohn, D. S., eds.), Bibl. Haemat. Vol. 43, pp. 457-459, Karger, Basel 136 Wyke, J. A. and Linial, M. (1973) Virology 53, 152-161 137 Hunter, E. and Vogt, P. K. (1976) Virology 69, 23-34 138 Stephenson, J. R., Reynolds, R. K. and Aaronson, S. A. (1972) Virology 48, 749-756 139 Stephenson, J R. and Aaronson, S. A. (1973) Virology 54, 53-59 140 Stephenson, J. R. and Aaronson, S. A. (1974) Virology 58, 294-297 141 Tronick, S. R., Stephenson, J. R., Verma, 1. M. and Aaronson, S. A. (1975) J. Virol. 16, 1476-1482 142 Friis, R. R. and Hunter, E. (1973) Virology 53, 479 143 Friis, R. R., Mason, W. M., Chen, Y. C. and Halpern, M. S. (1975) Virology 64, 49 144 Hanafusa, H, and Hanafusa, T. (1971) Virology 43, 313 145 Kawai, S. and Hanafusa, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3493 146 Robinson, W. S. and Robinson, H. L. (1971) Virology 44, 457-462 147 Hartley, J. W. and Rowe, W. P. (1966) Proc. Natl. Acad. Sci. U.S. 55, 78 148 Huebner, R. J., Hartley, J. W., Rowe, W. P., Lane, W. T. and Capps, W. I. 0966) Proc. Natl. Acad. Sci. U.S. 56, 1164 149 Maisel, J., Scolnick, E. M. and Duesberg, P. (1975) J. Virol. 16, 749 150 Maisel, J., Klement, W., Lai, M. M. C., Ostertag, W. and Duesberg, P. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3536 151 Beemon, K., Faras, A. J., Haase, A. T., Duesberg, P. and Maisel, J. E. (1976) J. Virol. 17, 525 152 Hu, S., Verma, 1. M., Dina, D. and Davidson, N. D., manuscript in preparation 153 May, J. T., Somers, K. D. and Kit, S. (1972) J. Gen. Virol. 16, 223-226 154 Peebles, P. T., Gerwin, B. I., PapaGeorge, A. G. and Smith S. 0975) Virology 67, 344 155 Bassin, R. H., Philips, L. A., Kramer, M. J., Haapala, D. K., Peebles, P. T. and Nomura, S. (1973) in Unifying Concepts of Leukemia (Dutcher, R. M. and Chieco-Bianchi, L., eds.), pp. 272-280, Karger, Basel 156 Somers, K. D., May, J. T., Kit, S., McCormick, K. J., Hatch, G. G., Stenbach, W. A. and Trentin, J. J. (1973) Intervirology 1, 11-18 157 Coffin, J. M. and Temin, H. M. 0971) J. Virol. 7, 625 158 Ross, J., Scolnick, E. M., Todaro, G. J. and Aaronson, S. A. (1972) Nat. New Biol. 231,163-167 159 Baltimore, D., McCaffrey, R. and Smoler, D. F. 0973) in Virus Research (Fox, C. F. and Robinson, W. S., eds.), pp. 51-59, Academic Press, New York 160 Yang, W. K., Kob, C. K. and Waters, L. C. (1972) Biochem. Biophys. Res. Commun. 47, 505 161 Mondal, H., Gallagher, R. E. and Gallo, R. C. (1975) Proc. Natl. Acad. Sci. U.S. 72, i194 162 Gerwin, B. I., Smith, S. J. and Peebles, P. T. (1975) Cell 6, 45 163 Coffin, J. M. and Temin, H. M. (1971) J. Virol. 8, 630 164 Altaner, C. and Temin, H. M. (1970) Virology 40, ll8 165 Linvingston, D. M., Serxner, L. E., Howk, D. J., Hudson, J. and Todaro, G. J. 0974) Proc. Natl. Acad. Sci. U.S. 71, 57 166 Kang, C. Y. and Temin, H. M. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1550 166(a) Mizutani, S. and Temin, H. M. (1973) J. Virol. 12, 440--448 167 Mayer, R. J., Smith, R. G. and Gallo, R. C. 0974) Science 185, 864

38 168 Benveniste, R. E., Lieber, M. M., Livingston, D. M., Sherr, C. J., Todaro, G. J. and Kater, S. S. (1974) Nature 248, 17 169 Schidovsky, G. and Ahmed, M. (1973) J. Natl. Cancer Inst. 51,225 170 Brown, D. D. and David, 1. B. (1968) Science 160, 272 17l Gall, J. G. (1968) Proc. Natl. Acad. Sci. U.S. 60, 553 172 Perkowska, E., MacGregor, H. L. and Birnsti~l, M. (1968) Nature 217, 649 173 Brown, D. D. and Blackler, A. W. (1972) J. Mol. Biol. 63, 75 174 Crippa, M. and Tocchini-Valentini, G. P. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2769 175 Tocchini-Valentini, G. P., Mahdavi, V., Brown, R. and Crippa, M. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 551 176 Brown, R. D. and Tocchini-Valentini, G. P. (1974) in Methods in Enzymology (Grossman, L. and Moldave, K., eds.), Vol. 29, pp. 173-177 177 Bird, A. Rogers, E. and Birnstiel, M. (1973) Nature 242, 226 178 Gallo, R. C. Yang, S. S. and Ting, R. C. (1970) Nature 228,927 179 Bhattacharyya, J., Xuma, M., Reitz, M., Sarin, P. S. and Gallo, R. C. (1973) Biochem. Biophys. Res. Commuo. 54, 324 180 Sarngadharan, M. G., Sarin, P. S,, Reitz, M. S. and Gallo, R. C. (1972) Nat. New Biol. 240, 67 181 MiJller, W. E. G., Yamazaki, Z. I., Zahn, R. K., Brehm, G. and Korting, G, (1971) Biochem. Biophys. Res. Commun. 44, 433 182 Schlom, J., Spiegelman, S. and Moore, D. (1972) Science 175, 542 183 Baxt, W., Hehlmann, R. and Spiegelman, S. (1972) Nat. New Biol. 240, 72 184 Axel, R., Schlom, J. and Spiegelman, S. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3133 185 Kuffe, D., Hehlmann, R. and Spiegelman, S. (1973) Proc. Natl. Acad. Sci, U.S. 70, 5 186 McCaffrey, R., Smoler, D. and Baltimore, D. (1973) Proc. Natl. Acad. Sci. U.S. 70, 521 187 Gerard, G. F., Rottman, F. and Green, M. (1974) Biochemistry 13, 1632 188 Rothenberg, E. and Baltimore, D. (1976) J. Virol. 17, 168 189 Collett, M. S. and Faras, A. J. (1975) J. Virol. 16, 1220 190 Junghans, R. P., Duesberg, P. H. and Knight, C. A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4895 191 Taylor, J. (1976) ICN-UCLA Syrup. Mol. Cell. Biol., Animal Virol. (Baltimore, D. and Huang, A.S., eds.), in the press 192 Verma, I. M., Temple, G. F., Fan, H. and Baltimore, D. (1972) Nat. New Biol. 235, 163-167 193 Kacian, D. L., Spiegelman, S., Bank, A., Terada, M., Metafora, S., Dow, L. and Marks, P. A. (1972) Nat. New Biol. 235, 167-169 194 Ross, J., Aviv, H., Scolnick, E. and Leder, P. (1972) Proc. Natl. Acad. Sci. U.S. 69, 264-268 195 Verma, I. M., Temple, G, F., Fan, H. and Baltimore, D. (1973) in Symposia on Control of Transcription, Calcutta, Inida (Biswas, B. B. et al., eds.)., pp. 355-372 196 Diggelmann, H., Faust, Jr., C. H. and Mach, B. (1973) Proc. Natl. Acad. Sci U.S. 70, 693-696 197 Verma, I. M., Firtel, R. A., Lodish, F, H. and Baltimore, D. (1974) Biochemistory 13, 3917 198 Berns, A. J. M., Blumenthal, H., Kaufman, S. and Verma, I. M. (1973) Biochem. Biophys. Res. Commun. 52, 1013-1019 199 Kacian, D. L, and Myers, J. C. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2191-2195 200 Maniatis, T., Kee, S. G., Efstratiadis, A. and Kafatos, F. C. (1976) Cell 8, 163-182 201 Efstratiadis, A., Maniatis, T., Katatos, F. C., Jeffrey, A. and Vournakis, J. N. (1975) Cell 4, 367 202 Taylor, J., Illmensee, R. and Summer, J. (1976) Biochim. Biophys. Acta, in the press 203 Hell, A., Young, B. D, and Birnie, G. D. (1976) Biochim. Biophys. Acta 442, 37-49 204 Tamblyn, T. M., and Wells, R. D. (1975) Biochemistry 14, 1412-1425