Vol. 22, No. 1 Printed in U.S.A.

JOURNAL OF VIROLOGY, Apr. 1977, p. 16-22 Copyright X 1977 American Society for Microbiology

RNA-Dependent DNA Polymerase Associated with Equine Infectious Anemia Virus1 BENEDICT G. ARCHER,* TIMOTHY B. CRAWFORD, TRAVIS C. McGUIRE, AND MARVIN E. FRAZIER Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164,* and Battelle Pacific Northwest Laboratory, Richland, Washington 99352

Received for publication 10 March 1976

Equine infectious anemia (EIAV) is shown to have an associated RNAinstructed DNA polymerase similar in its cofactor requirements and reaction conditions to the RNA tumor virus DNA polymerases. Demonstrating this DNA polymerase activity requires a critical concentration of a nonionic detergent, all four deoxyribonucleoside triphosphates, and a divalent metal ion. The reaction is sensitive to RNase, and a substantial fraction of the DNA synthesized is complementary to viral RNA. The detection of a complex of tritium-labeled polymerase product DNA-template RNA, which sedimented at 60S to 70S, provided evidence that EIAV contains high-molecular-weight RNA. These results, obtained with both virus propagated in cell culture and virus from the serum of an experimentally infected horse, indicate that EIAV may properly be considered a member of the family Retroviridae. They may also be pertinent to the mechanism(s) of viral persistence and periodic recrudescence of disease in chronically infected horses. Equine infectious anemia virus (EIAV) EIAV is an RNA-containing virus (16). The causes a natural disease of horses that is char- inhibition of the replication of EIAV by 5-iodoacterized by irregularly recurring episodes of deoxyuridine added to culture systems shortly pyrexia, weight loss, and anemia (11) for a after infection indicates that DNA synthesis is variable period after initial infection. Most sur- required for its replication (9). These distinctive viving horses eventually become asympto- properties of EIAV provided the motivation for matic, but virus or virus-antibody complexes the experiments reported here, which have de(10) are detectable in the sera ofthese horses for tected an RNA-dependent DNA polymerase aslife. Some of the lesions have an immunopatho- sociated with EIAV. We also present evidence genetic origin. The anemia is associated with that the EIAV RNA genome is of high molecuC3-coated erythrocytes (12), and a glomerulone- lar weight. phritis is caused by the deposition of immune MATERIALS AND METHODS complexes. Several characteristics of EIAV have Virus. The Wyoming strain of EIAV, adapted to prompted the suggestion (10) that EIAV may be equine fibroblasts (13), was obtained from W. related to the RNA tumor virus group (family Malmquist, National Animal Disease Laboratory, Retroviridae [31). Like most RNA tumor vi- Ames, Iowa. The virus was propagated in rollercultures of equine fibroblasts (E. Derm, CCLruses, EIAV is capable of initiating an in vitro bottle and used at passages 75 to 85. In vivo propagated 57) noncytocidal persistent infection of diploid cells virus was by collecting plasma from a from its natural host (9). It has been shown to horse that obtained had been inoculated 8 days previously be approximately spherical, with a diameter of with 10 ml of plasma from a horse in the acute stage 80 to 120 nm, to acquire an envelope during of experimental infection with the Wyoming strain maturation by budding from the plasma mem- of EIAV. The titer of the plasma was 107-5 50% brane, and to contain a nucleoid of about 50 nm infective doses/ml as determined in horse leukocyte (14, 20). Two types of cores have been observed cultures (9). Radioimmunoassay for viral antigen. Purified in the virions, suggestive of mature and immawhole EIAV was disrupted with ether and labeled ture C-type particles. Studies of EIAV in pri- with using chloramine T. The labeled proteins mary horse leukocyte cultures with labeled pre- were 125I fractionated on Bio-Gel P-100 after removing cursors of RNA and DNA have established that free [125I]Na on a Sephadex G-25 column. A fraction containing a viral protein of approximately 26,000 molecular weight, which formed a line of identity

1 Paper no. 4743, Experiment Station Project 0146, U. S. Department of Agriculture.

16

VOL. 22, 1977

EQUINE INFECTIOUS ANEMIA VIRUS DNA POLYMERASE

with an antigen ofthe same size that can be purified from the spleen of an infected horse (15, 17), was used for radioimmunoassay. Antiserum against EIAV was produced in rabbits against purified, ether-treated EIAV. Goat antiserum against rabbit gamma globulin was produced using standard procedures. With these reagents, a double-antibody competitive radioimmunoassay for EIAV antigen was performed, essentially according to Hunter (6). A complete description of this procedure will be included in a report of a comparative study of EIAV antigens (manuscript in preparation). Virus purification. EIAV was precipitated from clarified culture medium by adding polyethylene glycol 6000 to a final concentration of 8%. The precipitated virus was centrifuged at 15,000 x g for 20 min, and the resulting pellet was resuspended in a volume of 0.01 M sodium phosphate, pH 7.5, 0.001 M EDTA equal to 1/30 of the initial volume. After clarification, this virus was resuspended and overlaid onto a 20 to 50% discontinuous gradient of sucrose in the same buffer and centrifuged at 82,000 x g for 2 h. The visible band at the 20 to 50% interface was collected, diluted twofold, and overlaid onto a linear 20 to 60% sucrose gradient, which was centrifuged for 14 h at 100,000 x g. The gradient was fractionated from the bottom of the tube, or in some cases the visible band was withdrawn from the top by pipette. More recently, in some experiments EIAV was concentrated from culture medium by centrifugation in 250-ml polypropylene bottles at an average force of 20,000 x g for 5 h. The pellets obtained were resuspended in 1/250 of the initial volume of 0.01 M sodium phosphate buffer, pH 7.5, and this resuspended virus was processed through the same discontinuous and continuous sucrose gradients described above. Protein determinations were done by the fluorescamine assay (2), using bovine serum albumin as a standard. Polymerase assay. A suspension of virus was prepared for DNA polymerase measurements by adding dithiothreitol and Triton X-100 to final respective concentrations of 0.02 M and 0.05 to 0.11%. After a few minutes in ice, samples of the detergent-treated virus were transferred to siliconized glass tubes, and a mixture of substrates and cofactors was added to give a reaction solution containing 0.02 to 0.06% Triton X-100, 0.01 M dithiothreitol, 0.08 M Tris (pH 7.9; 7.5 at 370C), 0.001 M each dATP, dCTP, and dGTP, 80 ACi of [3H]TTP (specific activity, 16.6 or 45 Ci/mmol) per ml, and 0.006 M magnesium chloride. Zero-time samples were withdrawn from the reaction mixtures immediately after adding the substrates. The reaction mixtures were incubated at 370C, and samples were withdrawn at timed intervals and added to 0.5 ml of cold 0.1 M sodium pyrophosphate. Each sample was precipitated with 50 Img of yeast RNA and 2 ml of cold 10% trichloroacetic acid. The precipitates were collected on glass-fiber or cellulose nitrate filters, washed three times with cold 0.2 M HCl, 0.02 M sodium pyrophosphate, dried, and counted at an efficiency of approximately 18% in 10 ml of toluene containing 2,5-diphenyloxazole (PPO) and 1,4-bis-(5-phenyloxozolyl)benzene (POPOP) fluors. To measure polymerase activity

17

without purifying the virus, 30 ml of medium from infected cultures or 30 ml of virus-containing plasma was clarified by low-speed centrifugation and then overlaid onto 7 ml of 30% glycerol in 0.15 M sodium chloride, 0.01 M Tris (pH 8.3), 0.002 M EDTA and centrifuged for 90 min at 82,000 x g. The pellets were resuspended in a small volume of 0.01 M Tris, pH 8.3; dithiothreitol and Triton X-100 were added to 0.02 M and 0.11%, respectively. The resuspended pellets were pooled and assayed as described above. The sensitivity of the reaction to RNase was tested by adding RNase A and T, to 80 and 15 ,ug/ml, respectively, and incubating for 45 min at 370C in the presence of 0.20 M sodium chloride. At the end of this incubation, the samples were transferred to ice for the addition of substrates and cofactors, which reduced the concentration of sodium chloride by dilution to 45 mM in the final polymerase reaction mixtures. Control reactions were identical except for the omission of the RNases. The ability of the DNA polymerase to utilize a synthetic primer-template was tested by adding oligodeoxythymidine[oligo(dT)I.polyribodenylic acid[poly(RA)I to a final concentration of 0.5 absorbency unit at 260 nm/ ml. In these cases all four nucleoside triphosphates or only TTP were present as noted below. The [3H]TTP was obtained from New England Nuclear Corp., unlabeled deoxynucleoside triphosphates, Triton X-100, and RNases were from Sigma Chemical Co., and oligo(dT) * poly(A) was from Miles Laboratories. RNA-DNA hybridization. A DNA polymerase reaction with 96 ,ug of purified viral proteins in a final reaction volume of 275 1,l was carried out as described above, except that the concentration of TTP (specific activity, 5.3 Ci/mmol) was increased to 0.05 mM and actinomycin D was added to a concentration of 40 jig/ml. After 3 h at 37°C, EDTA, sodium chloride, sodium dodecyl sulfate, and salmon sperm DNA were added to 13 mM, 100 mM, 0.8%, and 35 jig/ml, respectively, and the mixture was extracted at room temperature twice with freshly distilled and neutralized phenol-metacresol (7:1, wt/wt) and twice with chloroform-isoamyl alcohol (99:1, vol/vol). Sodium acetate (pH 5.0) was added to the aqueous phase to a concentration of 0.1 M, and the polynucleic acids were precipitated with 2 volumes of ethanol at -20°C for 12 h. The precipitate was recovered by centrifugation at 12,000 x g for 15 min, redissolved in 0.5 ml of 0.5 M sodium hydroxide, and held at 40°C to hydrolyze RNA. After 1 h at this temperature, 0.8 ml of 0.25 M acetic acid was added, followed by precipitation of the nucleic acids with ethanol. To obtain viral RNA for the hybridization experiment, an amount of purified virus containing approximately 0.5 mg of viral proteins was pelleted and resuspended in 1 ml of 0.01 M Tris (pH 7.5)-0.1 M sodium chloride-1.0 mM EDTA-0.2% diethylpyrocarbonate. Purified yeast RNA, sodium dodecyl sulfate, and self-digested Pronase were added to 0.4 mg/ml, 0.5%, and 0.25 mg/ml, respectively, and the mixture was incubated at 370C for 30 min. The RNA was then isolated and precipitated as described above for the extraction of the products of the polym-

18

ARCHER ET AL.

erase reaction. Both the precipitated RNA and the [3H]DNA product were dried under vacuum and redissolved together in 150 1.l of 0.3 M sodium chloride-0.05 M Tris (pH 7.3)-0.1% sodium dodecyl sulfate-1.0 mM EDTA-2% phenol. A 50-/tl portion of the mixture was sealed in a siliconized capillary tube and held at 630C for 18 h. An identical capillary tube was kept at -20'C. At the end of the incubation period the contents of the incubated capillary and one-half the contents of the control capillary were mixed with 4.8-ml volumes of Cs2SO4 in 0.01 Tris (pH 7.3)-5 mM EDTA. The starting density of the solutions was 1.55 g/ml, and centrifugation was at 33,000 rpm and 18'C for 68 h in an SW50L rotor. Fractions were collected by bottom puncture, precipitated with yeast RNA and 10% trichloroacetic acid, and counted on cellulose nitrate filters. Rate-zonal sedimentation analysis of labeled nucleic acids. The procedure of Schlom and Spiegelman (18) for the simultaneous detection assay was used. Viral polymerase reactions were performed as described above, except that both [3H]TTP (specific activity, 19 Ci/mmol) and [3H]dGTP (specific activity, 25 Ci/mmol) were present at 1.4 x 10-4 and 4.7 x 10-5 M, respectively, actinomycin D was added at 50 tLg/ml, and the total volume was increased to 0.2 ml. The reactions were stopped after 15 min by adding sodium chloride and sodium dodecyl sulfate to 0.2 M and 0.5%, respectively, and the mixtures were shaken gently with 1 to 2 ml of phenol-cresol. Threetenths-milliliter volumes of the aqueous phases of the extraction mixtures were overlaid onto 12.5-ml 10 to 30% glycerol gradients in 0.15 M sodium chloride, 0.01 M Tris (pH 8.3), 0.002 M EDTA and centrifuged at 10°C for 3 h at 193,000 x g. The entire gradients were fractionated by piercing the tube bottoms, and each fraction was tested for trichloroacetic acid-precipitable radioactivity. 3H-labeled mammalian rRNA's, purchased from Schwarz/ Mann, were used as markers in a separate gradient.

RESULTS Characterization of the enzyme reaction. The synthesis of polymeric DNA by the endogenous EIAV DNA polymerase reaction is illustrated in Fig. 1. A high initial rate of synthesis slowed by about 30 min to a fairly constant rate, which continued beyond 4 h. The addition of actinomycin D to the reaction mixture reduced the synthesis of DNA by approximately 50%. A specific activity of 460 pmol of dTMP incorporated per mg of viral protein in 60 min can be calculated from the data in Fig. 1. The addition of the synthetic primer-template oligo(dT) . poly(rA) increased the reaction rate 50- to 150-fold (Table 1). Essentially no activity was detected in the medium from cell cultures not infected with EIAV, even when a synthetic primer-template was added. In virus pelleted from the plasma taken from horses 8 days after inoculation with EIAV, low but reproducible levels of DNA polymerase activity were detect-

J. VIROL.

E

0

30

60

0o

120

minutes

150

180

FIG. 1. Kinetics of DNA synthesis by EIAV-associated DNA polymerase. A 20-pil portion containing 0.8 pg of viral protein was withdrawn at the indicated times from a control reaction (@). Actinomycin D (50 Mg/ml) was present in a parallel reaction (0).

able without the addition of an exogenous primer-template. Easily detectable activity was observed when oligo(dT) poly(rA) was supplied. No activity was found in the preinoculation plasma from the same horses. For the reaction using only the endogenous viral RNA as primer-template, all four triphosphate deoxynucleosides were required. Deletion of any one reduced the polymerase activity by more than 70% (Table 2). Only a slight decrease in activity resulted from omitting the reducing agent dithiothreitol (data not shown). Incubation of the disrupted virus with RNase in the presence of 0.20 M sodium chloride produced an 84% decrease in the amount of DNA synthesized (Table 3). A divalent cation, either magnesium or manganese, was needed for activity (Fig. 2A). The maximum rate attainable in the endogenous reaction with magnesium as a cofactor is greater than that achieved with manganese, and it occurs at a concentration of about 5 ml. The activity with manganese as the cofactor peaks sharply at a concentration of about 1 mM, and higher concentrations are inhibitory, perhaps activating a competing process. Whereas higher concentrations of magnesium (up to 25 mM) are also inhibitory, the inhibition is less than that caused by excess manganese. The effect of pH on endogenous reaction rates is shown in Fig. 2B, where it is evident that the optimal pH (corrected to 37°C) is approximately 7.6. Treatment of the virus with a nonionic detergent was required for full activity. The optimal concentration of Triton with purified virus was about 0.02% (Fig. 2C), and higher concentrations were inhibitory. The optimal concentration of detergent was variable when determined with unpurified virus. -

EQUINE INFECTIOUS ANEMIA VIRUS DNA POLYMERASE

VOL. 22, 1977

TABLE 1. Comparison ofRNA-dependent DNA polymerase activity with endogenous and exogenous

primer-templates [3H]TMP incorpo-

Virus

Primer-template

Tissue culture Tissue culture Uninfected tissue culture Infected equine plasma Infected equine plasma Preinoculation equine plasma

Endogenous Oligo(dT) poly(rA) Oligo(dT) poly(rA) Endogenous

rated during 60 mi (cpm) 4,732 674,714 142

442

Oligo(dT) poly(rA)

18,975

Oligo(dT) poly(rA)

224

a This reaction mixture contained only one nucleoside triphosphate, ¶TP; all others contained a complete complement of DNA precursors.

Association of the enzyme with the EIA virion. To demonstrate that the DNA polymerase is associated with the virus particle, purified EIAV was centrifuged overnight at 100,000 x g in a 20 to 60% continuous gradient of sucrose, which was then fractionated and tested for both EIA viral antigen and polymerase activity. It can be seen in Fig. 3 that the viral antigen and the endogenous polymerase activity are confined to coincident peaks centered at a density of about 1.15 g/ml. In some experiments, DNA polymerase activity measured with an exogenous template was detected higher in the tube (lower density) than viral antigen. RNA-DNA hybridization. The results of a qualitative annealing experiment, designed to determine if the DNA product of the polymerase reaction was transcribed from the viral RNA, are shown in Fig. 4. Incubation of viral RNA with DNA purified from a polymerase reaction caused approximately 45% of the labeled DNA to migrate in an equilibrium cesium sulfate gradient to densities higher than is characteristic for DNA. In an identical mixture of RNA and polymerase product, which was kept at -20°C during the incubation period, all the radioactivity was present in a band at a density appropriate for DNA. Analysis of labeled nucleic acids in a reaction mixture. EIAV that was concentrated by precipitation with polyethylene glycol and isopycnically banded in sucrose gradients was subjected to the simultaneous detection (18) procedure. The nucleic acids from reactions carried out with this purified virus were extracted with phenol-cresol and analyzed by rate-zonal sedi-

19

mentation on 10% to 30% glycerol gradients. Labeled material that sedimented at 60S and 70S was repeatably detected in four experiments (Fig. 5). If the disrupted virus was incubated with RNase before the reaction was carried out, no rapidly sedimenting material was present. Fast-sedimenting, labeled material was also detectable in reactions carried out with pelleted, but otherwise unpurified, virus from both cell cultures and equine plasma.

DISCUSSION The data in this report demonstrate the presence of an RNA-dependent DNA polymerase associated with the EIA virion. An approximately 10-fold enhancement in endogenous activity results from treatment ofthe virus with a nonionic detergent, indicating that disruption of the viral envelope promotes the DNA polymerase reaction. The dependence of the DNA polymerase activity on the concentration of detergent used to disrupt the virus is similar to that reported for both murine and avian RNA tumor viruses in that both are optimally activated by a relatively low concentration of detergent and higher concentrations are inhibitory (7, 19). This requirement for the disruption of the virus by detergent, and the observation that the enzyme activity remains associated with the virus in an amount proportional to the amount of virus (Fig. 3) through rate-zonal and isopycnic centrifugation purification procedures, are consistent with the interpretation that the DNA polymerase activity is a component of EIAV. The experiment that demonstrates that a substantial fraction of the DNA polymerase product will form a DNA-RNA hyTABLE 2. Requirements of the endogenous EIAV RNA-dependent DNA polymerase reaction Reaction mixture

Complete dATP omitted dCTP omitted dGTP omitted dATP and dGTP omitted

[3HlTMP incorporated during 60 min (cpm)

2,627 760 788 432 344

TABLE 3. Sensitivity of the EIAV RNA-dependent DNA polymerase reaction to preincubation with RNase Reaction mixture

Control Preincubated with RNase

[3HlTMP incorporated during 60 min (cpm)

4,890 806

20

ARCHER ET AL.

J. VIROL. Il

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B -D

0 0

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o

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'o x

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2-

, 0.6

*0

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0 L L.

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0~

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7.5

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0 x

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FIG. 2. Dependence ofpolymerase activity on magnesium (0) and manganese (a) (A), pH (B), and Triton X-100 concentration (C). The reactions in (A) were done as described with unpurified virus. Each point in (B) is the average value of four experiments with purified virus. Each point in (C) represents the DNA synthesized by 0.7 Mg of purified viral proteins.

brid with viral RNA indicates that the viral RNA is functioning as a template in the DNA polymerase reaction. The residual polymerase activity that remains when one or two nucleoside triphosphate precursors are omitted (Table

2) is explained by contamination of the sources of those nucleosides which are added with traces of the one or two omitted, or traces of the deleted precursors in the virus preparation itself, as these experiments were done with un-

VOL. 22, 1977

EQUINE INFECTIOUS ANEMIA VIRUS DNA POLYMERASE

1 25 ,

An

-

I

1.10.

3.0-

.09

2.0 l, al 1 - °e

2.0 -

S E C

s/

1.0

5

ia3

20ft ~15nnber20

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FIG. 3. Association of DNA polymerase with EIAV antigen. Endogenous DNA polymerase activity (0) and EIAV antigen (@) were measured as scribed on fractions from a 20 to 60% sucrose gradient of a 10-ml total volume. Densities were measured by weighing 100-p samples in capillary pi-

pettes.

9000

-_

purified virus. The residual activity that remains after preincubation with RNase (Table 3) is explainable by the fact that the incubation with RNase is in the presence of high salt to ensure that the primer is not dissociated from the template, but the salt also has the effect of stabilizing the secondary structure of the RNA and reducing its susceptibility to hydrolysis by RNases. In addition to having an RNA-instructed DNA polymerase with properties similar to those of the analogous enzymes from the RNA tumor viruses (4), the detection of labeled nucleic acids sedimenting at roughly 608 to 708 in the simultaneous assay procedure indicates that the virion contains high-molecular-weight RNA. These two characteristics, together with morphological resemblance of EIAV to the RNA tumor viruses (20) and the fact that the major protein component of EIAV has an apparent molecular weight of approximately 26,000 (15, 17; B. G. Archer, unpublished data), establish that EIAV may properly be classified with the Retroviridae. Several aspects of the life cycle of EIAV in horses are unusual, especially the lifelong presistence of the virus with irregularly recurring the larence large creases iin the tith titer ofirculatingevirus circulating virus accompanied by acute clinical signs. Different proposed explanations of this disease pattern invoke interactions among the various components of the immune system (5, 11), or the possibility of a periodic shift in the antigenic

1A

_

40.0 70S 8700oiD 700-69ot

iX

\

21

j

60S

28S 18$

4S

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FIG. 4. Hybridization of polymerase product [3H]DNA to purified unlabeled EIAV RNA. The open circles represent data obtained from a mixture ofPHIDNA and RNA, which was incubated at 630C for 18 h. A control mixture, which was held at -200C during the incubation period, is represented by the solid circles. Densities were calculated from measured refractive indexes.

FIG. 5. Analysis by rate-zonal sedimentation of the labeled nucleic acids from EIAV DNA polymerase reaction mixtures. Nucleic acids were extracted from a control reaction (0) carried out as described and from another (0) that was preincubated for 45 min at 370C with RNase A, RNase T,, and sodium chloride.

22

composition of the viral envelope (8). If it can be demonstrated that the replication of EIAV involves an integrated DNA provirus, as is the case for other retroviruses, then the significance of this fact to the persistence of the virus in the natural disease will have to be considered. ACKNOWLEDGMENTS We thank W. Malmquist for the initial stock of virus, and J. M. Bishop for helpful suggestions and discussion. This work was supported by Public Health Service grants AI07471 and FR5465 from the National Institute of Allergy and Infectious Diseases and the Division of Research Facilities and Resources, respectively, and by U. S. Department of Agriculture Cooperative Agreement 12-14100-9067.

ADDENDUM IN PROOF A report (Charman et al., J. Virol. 19:1073-1079, 1976) appearing after the present article had been submitted described exogenous reverse transcriptase activity in EIAV in the presence of synthetic primer templates. 1.

2. 3.

4.

5. 6.

7.

J. VIROL.

ARCHER ET AL.

LITERATURE CITED Banks, K. L., J. B. Henson, and T. C. McGuire. 1972. Immunologically mediated glomerulitis of horses. I. Pathogenesis of persistent infection by equine infectious anemia virus. Lab. Invest. 26:701-707. Bohlen, P., S. Stein, W. Dairman, and S. Udenfriend. 1973. Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 1551:213-220. Fenner, F. 1976. The classification and nomenclature of viruses. Summary of results of meetings of the International Committee on Taxonomy of Viruses in Madrid, September 1975. Virology 71:371-378. Green, M., and G. F. Gerard. 1974. RNA-directed DNA polymerase-properties and functions in oncogenic RNA viruses and cells. Prog. Nucleic Acid Res. Mol. Biol. 14:187-334. Kono, Y. 1969. Viremia and immunological responses in horses infected with equine infectious anemia virus. Natl. Inst. Anim. Health Q. 9:1-9. Hunter, W. M. 1967. The preparation of radioiodinated proteins of high activity, their reaction with antibody in vitro: the radioimmunoassay, p. 608-642. In D. M. Weir (ed.), Handbook of experimental immunology. F. A. Davis Co., Philadelphia. Junghans, R. P., P. H. Duesberg, and C. A. Knight.

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1975. In vitro synthesis of full-length transcripts of Rous sarcoma virus RNA by viral DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 72:4895-4899. Kono, Y., K. Kobayashi, and Y. Fukunaga. 1973. Antigenic drift of equine infectious anemia virus in chronically infected horses. Arch. Gesamte Virusforsch. 41:1-10. Kono, Y., T. Yoshino, and Y. Fukanaga. 1970. Growth characteristics of equine infectious anemia virus in horse leukocyte cultures. Arch. Gesamte Virusforsch. 30:252-256. McGuire, T. C., T. B. Crawford, and J. B. Henson. 1972. Equine infectious anemia: detection of infectious virus-antibody complexes in the serum. Immunol. Commun. 1:545-551. McGuire, T. C., and J. B. Henson. 1973. Equine infectious anemia-pathogenesis of persistent viral infection. Perspect. Virol. 8:229-247. McGuire, T. C., J. B. Henson, and D. Burger. 1969. Complement(C'3)-coated red blood cells following infection with the virus of equine infectious anemia. J. Immunol. 103:293-299. Malmquist, W. A., D. Barnett, and C. S. Becvar. 1973. Production of equine infectious anemia antigen in a persistently infected cell line. Arch. Gesamte Virusforsch. 42:361-370. Matheka, H. D., L. Coggins, J. N. Shively, and N. L. Norcross. 1976. Purification and characterization of equine infectious anemia virus. Arch. Virol. 51:107114. Nakajima, H., N. L. Norcross, and L. Coggins. 1972. Demonstration of antigenic identity between purified equine infectious anemia virus and an antigen extracted from infected horse spleen. Infect. Immu. 6:416-417. Nakajima, H., S. Tanaka, and C. Ushimi. 1970. Physicochemical studies of equine infectious anemia virus. IV. Determination of the nucleic acid type in virus. Arch. Gesamte Virusforsch. 30:273-280. Norcross, N. L., and L. Coggins. 1971. Characterization of an equine infectious anemia antigen extracted from infected horse spleen tissue. Infect. Immun. 4:528-531. Schlom, K., and S. Spiegelman. 1971. Simultaneous detection of reverse transcriptase and high molecular weight RNA unique to oncogenic RNA viruses. Science 174:840-843. Scolnick, E. M., S. A. Aaronson, and G. J. Todaro. 1970. DNA synthesis by RNA-containing tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 67:1034-1041. Tajima, M., H. Nakajima, and Y. Ito. 1969. Electron microscopy of equine infectious anemia virus. J. Virol. 4:521-527.