VIROLOGY

84, 75-86 (1978)

Ultraviolet

Transcriptional Unit Mapping for the Late Genes in Adenovirus Type 2

SHELDON Departments

of Biology

C. GIRVITZ* and Radiology,

AND ANDREW McMaster

University,

J. RAINBOW’ Hamilton,

Ontario,

Canada

Accepted August 16,1977

Human KB cells lytically infected with human adenovirus type 2 were pulse labeled with [““Slmethionine during the late period of viral infection, and the resulting labeled polypeptides were analyzed by SDS-polyacrylamide-gel electrophoresis and autoradiography. A differential reduction of late adenovirus type 2 protein synthesis was observed following uv irradiation of the infected cells. The uv inactivation of the synthesis of specific viral proteins was assumed to correlate with physical distance between the genes encoding these proteins and their promotors. In this way the relative positions of several late adenovirus type 2 gene locations were determined within each of two transcription units. INTRODUCTION

been determined (Tal et al., 1974; Craig et Human adenovirus type 2 (Ad 2) is one al., 1974; Philipson et al., 1974; Sharp et of the best characterized among the 31 al., 1974; Pettersson et al., 1976) giving human serotypes. Within an icosahedral rise to a transcriptional map for both early capsid, adenoviruses contain a double- and late cytoplasmic Ad 2 mRNA. Approximately 18 virus-specific proteins stranded linear DNA molecule of 23 x 10” daltons, which is capable of coding for have been identified in lytically infected about 20 average-size proteins (Green, cells (Anderson et al., 1973) and many of 1967). Upon entry into the nucleus, ade- these polypeptides have also been identinovirus DNA is transcribed into heteroge- fied in cell-free translation systems using neous nuclear RNA by a host RNA polym- polysomal RNA from lytically infected erase (Wallace and Kates, 1972; Price and cells (Anderson et al., 1974; Lewis et al., 1974; Westphal et al., 1974; Oberg et al., Penman, 1972). Following transcription 1975). Using hybridization techniques, the viral RNA is cleaved and transported mRNAs transcribed from specific regions to the cytoplasm and translated into virusspecific polypeptides. By fractionation on of the viral DNA complementary to DNA sucrose gradients, several discrete size restriction endonuclease fragments have classes of viral mRNA have been demon- also been isolated and the corresponding products strated and correlated with their poly- viral polypeptide translation peptide products (Anderson et al., 1974). have been synthesized in a cell-free system These transcription products have also (Lewis et al., 1975; Atkins et al., 1975). In been characterized according to their elec- this way groups of late viral genes corretrophoretic mobility (Tal et al., 1974;Craig sponding to each of the specific EcoRI, et al., 1974). By means of restriction endo- Bum1 and HpaI fragments of Ad 2 DNA have been determined. This information nucleases and hybridization techniques, the transcription of mRNA homologous to has led to a preliminary map for the localspecific regions of the Ad 2 genome has ization of the genes encoding late Ad 2 polypeptides. Lewis and co-workers (1975) have found that genes IIIa, III (penton), * Research Student of the National Cancer InstiIVa,, V (minor core), pVI1 (major core tute of Canada. ’ Author to whom reprint requests should be precursor), and IX occur on the endonucleaddressed. ase EcoRI-A fragment, which encompasses 75 0042-6822/78/0841-0075$02.00/O Copyright 0 1978by Academic Press, Inc. All rights of reproduction in any form reserved.

76

GIRVITZ

AND

about 60% on the left-hand end of the viral genome and that genes II (hexon) and pV1 are at the boundary between the EcoRI-A and EcoRI-B fragments, whereas genes 1OOKand IV (fiber) have been found to occur within the right-hand 30% of the viral genome. In this study we have employed the technique of uv transcription unit mapping to obtain a more detailed map of the gene positions for the late proteins on the DNA of Ad 2. This technique has been successfully applied in prokaryotic systems (Brautigan and Sauerbier, 1973;Hercules and Sauerbier, 1973) and also for the single-stranded vesicular stomatitis virus RNA (Ball and White, 1976) (for a detailed review see Sauerbier, 1975, 1976.) Exposure of Ad 2 to uv radiation results in many different photolesions in the viral DNA (Rainbow and Mak, 1973), some of which, if unrepaired, may result in the premature termination of transcription as has been found following uv irradiation of bacteriophage (Sauerbier et al., 1970) and of calf thymus DNA (Hagen et al., 1970). Consequently, for each transcription unit, uv irradiation of Ad 2 DNA would result in a preferential inhibition of transcription for the promotor-distal portions of the transcript resulting in shorter than normal RNA molecules. The length of these newly synthesized RNA chains would be equal to the distance between the promotor and the transcription-terminating lesion. Assuming a random distribution of transcription-terminating lesions, the probability that any given gene will be expressed is thus inversely proportional to the distance along the DNA between the promotor of transcription and the termination of the gene. This relation would be reflected in a differential reduction of protein synthesis for the various Ad 2 proteins. In this article we report studies using SDS-polyacrylamide-gel electrophoresis to determine the uv-inactivation curves for the synthesis of the majority of late Ad 2 proteins. Based on the preliminary map determined by Lewis and co-workers (1975) together with the assignment of the majority of late Ad 2 genes to the light

RAINBOW

strand (Oberg, Mak, and Philipson, personal communication) our results suggest that for the light strand of the Ad 2 genome there are two transcriptional units which code for the majority of the Ad 2 late proteins. By assuming that the ultraviolet sensitivity of the synthesis of each protein is correlated with the distance between the promotor and the 5’-terminus of the gene encoding that protein, we have been able to order some of the genes previously mapped to the EcoRI fragments of the Ad 2 genome. MATERIALS

AND

METHODS

Cells and virus. Human KB cells were grown on 60-mm petri dishes (Falcon Plastics, Oxnard, Calif.) in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (10 pg/ml), and fungizone (2.5 pug/ml) (Grand Island Biological Co., Grand Island, N.Y.). Virus stock was prepared by infecting KB cells in suspension and was subsequently purified by the method of Green and Pina (1963). The virus band collected from the second cesium chloride gradient was diluted lo-fold with TBS (Winocour, 1963) plus 20% glycerol and was stored at -70” until use. Storage under these conditions produced no significant loss of infectivity. Ad 2 stocks prepared in this manner had titers of 2.5 x lOlo to 5.0 x lOlo PFU/ml. Virus infection, uv irradiation, and preparation of [%‘lmethionine-labeled cell extracts. Confluent 60-mm plates of

about 3.5 x 10” KB cells were infected at 50 PFU of Ad 2 per cell in an inoculum of 0.2 ml of phosphate-buffered saline (PBS). Mock infections were carried out with 0.2 ml of PBS only. Viral adsorption was continued for 1 hr at 37” with periodic gentle rocking of the dishes to spread the viral inoculum over the monolayer. Following adsorption, 5 ml of fresh warm MEM supplemented with 10% fetal calf serum was added. At an appropriate time after infection, usually 10 hr, the medium was removed and the infected cells were washed once with PBS. The infected KB monolayer was then exposed to ultraviolet (uv) irra-

UV TRANSCRIPTION

UNIT

diation of 254 nm. Dosimetry of the uv germicidal lamp (General Electric G8T) was carried out using a J-225 short wave uv meter (Ultra-Violet Products, Inc., San Gabriel, Calif.). The incident dose rate employed under these conditions was 10 to 50 ergslmm’lsec with doses ranging from 0 to 250 ergs/mm’. At an appropriate time after irradiation, usually 6 hr, the polypeptides of mockinfected cells and virus-infected cells were labeled with [3”S]methionine. Immediately before labeling, the medium of each plate was removed and replaced with 2.7 ml of methionine-free MEM containing 10% fetal calf serum. This was followed by the addition of 0.3 ml of normal MEM supplemented with the desired amount of 13”Slmethionine (AmershamlSearle, Arlington Heights, Ill.). Usually, 35 PCi of [3”Slmethionine with a specific activity of 400 Ci/mmol was added per plate (3 Kg of methionine/ml). Under these conditions the uptake of [“Slmethionine remained linear beyond the l-hr labeling period. Under similar conditions, a linear uptake has also been reported by other workers (Weber, 1976). After a 1-hr pulse, Ad 2infected cells were harvested from the plates with a rubber policeman and were added to an equal volume of crushed frozen PBS. Cells were collected by centrifugation at 1000 g for 5 min at 4”. The cell pellets were washed twice by suspension in cold PBS and resuspended in 100 ~1 of cold PBS. Samples were frozen at -70” or immediately processed for electrophoresis as described below. [““SlMethionine-labeled adenovirus for virion component comparison was a generous gift from N. Lassam, McMaster University. Infected cell cultures were labeled with [““Slmethionine from 16 to 30 hr postinfection, and were harvested by centrifugation at 48 hr postinfection. Purification of adenovirus was carried out as described above. Sodium dodecyl sulphate (SDS)-polyacrylamide-gel electrophoresis. SDS-poly-

acrylamide gels used for analytical purposes as described by Laemmli (1970) were formed as slabs 1.5 mm thick between glass plates. A stacking gel of 5.0% acryl-

MAPPING

IN Ad 2

77

amide and 0.16% bisacrylamide containing 12 sample slots was cast above the running gel of 15% acrylamide and 0.86% bisacrylamide. Samples were prepared for electrophoresis by disruption of 10 ~1 of sample with an equal volume of 2x SDS sample buffer (1 x SDS sample buffer-O.0625 M Tris, pH 6.8; 2% SDS (Serva); 10% glycerol; 0.001% bromophenol blue; 0.1 M dithiothreitol) as described by Anderson and Gesteland (1972). Prepared samples were heated in a water bath at 100” for 2 min and then 15 ~1 of sample were loaded in each slot. Electrophoresis was carried out at 80 to 120 V for 12 to 14 hr with the Tris-glycine-SDS electrophoresis buffer of Laemmli (1970). After electrophoresis, the gels were stained with 0.05% Coomassie brilliant blue (in 25% isopropanol, 6.5% acetic acid), destained, and dried under vacuum. Autoradiograms were exposed for 24 to 48 hr on Kodak RPR medical X-ray film. Under these conditions all film densities to be recorded, including background, fell on the linear part of the characteristic curve of the film. In some experiments, the autoradiogram was used as a guide for the excision of the appropriate radioactive polypeptide bands from the dehydrated gel. The dry gel fragments were dissolved in 0.2 ml of 30% H,O, at 37” for 24 hr, mixed with 10 ml of Aquasol (New England Nuclear), and analyzed by liquid scintillation counting. In other experiments, the autoradiograms were scanned using a microdensitometer (Joyce-Loebl). The relative amount of radioactive protein in each band was then determined from the area under each peak of the scan using a planimeter. RESULTS

Time Course of Synthesis of Late Viral Proteins in Infected KB Cells

The induction of late Ad 2 proteins is dependent on the initiation of viral DNA synthesis, which occurs at approximately 5 to 6 hr postinfection (Green et al., 1970). Consequently, in order to detect the effects of uv irradiation on the synthesis of late viral proteins it is necessary to irradiate the infected cells after the initiation of

78

GIRVITZ AND RAINBOW

DNA synthesis, but before large amounts of late viral-specific polypeptides are produced. It was also considered of importance to determine the time of [3”Slmethionine labeling which would permit the detection of sufficient synthesis of all the late Ad 2 polypeptides to be examined following uv irradiation of the infected cells. KB cells were infected at 50 PFU/cell with Ad 2 and were subsequently irradiated at a dose of 200 ergs/mm” at 8 hr postinfection. Similar nonirradiated infected cultures were kept as controls. The infected cells were then pulse labeled for 1 hr with 13”Slmethionine at various times after irradiation. The amounts of the different late Ad 2 polypeptides synthesized were determined by SDS-polyacrylamidegel electrophoresis. Autoradiograms displayed a variety of easily detectable late Ad 2 polypeptides similar to those reported previously (Anderson et al., 1975; Lewis et al., 1975; Weber, 1976). Typical results for the time course of polypeptide synthesis are shown in Fig. 1 for hexon, the gene II product. It can be seen that, for the nonirradiated culture, synthesis of Ad 2 hexon polypeptide was detectable at about 12 hr postinfection. Late virus-induced polypeptide synthesis continued until at least 24 hr postinfection, at which point the time course was terminated. This is consistent with previous reports indicating that most of the capsid antigens are detectable by 10 to 12 hr after infection (White et al., 1969; Lewis et al., 1975). It can also be seen that irradiation of the infected cells resulted in a considerable reduction in the synthesis of the hexon protein for all the times examined. A similar time course was found for the other late Ad 2 polypeptides (not shown). Since no late Ad 2 polypeptides could be detected before 12 hr after infection, the reduction in protein synthesis which resulted from the uv irradiation at 8 hr postinfection is thought to result from damage to the viral DNA rather than from damage to preexisting viral-specific mRNA. The uv inactivation of viral polypeptide synthesis is, therefore, thought to result from the induction of transcription-terminating le-

sions in the viral DNA. The fact that this radiation-induced depression of polypeptide synthesis was reduced at later times after irradiation suggests that at least some of these transcription-terminating lesions can be repaired. Differential uv Inactivation Polypeptide Synthesis

of Late Ad 2

In order to determine the uv inactivation for the synthesis of the different late Ad 2 polypeptides, Ad 2-infected KB cells were uv irradiated with a series of increasing doses at 10 hr postinfection and were pulse labeled for 1 hr at 16 hr postinfection. An equal volume of each sample extract, obtained from an equal number of infected cells, was then analyzed by SDS-polyacrylamide-gel electrophoresis and autoradiography. A typical autoradiogram is shown in Fig. 2. It can be seen that increasing uv doses to the infected cells result in a differential reduction in polypeptide synthesis for the different late

TIME

AFTER

INFECTION

(HOURS)

FIG. 1. Time course for the detection of the hexon protein. Ad Z-infected KB cells from cultures not irradiated and from cultures uv irradiated at 8 hr postinfection with 200 ergs/mm* were pulse labeled for 1 hr with [YS]methionine at different times after infection. These samples were processed as described in the text and subjected to SDS-polyacrylamide-gel electrophoresis and autoradiography. The hexon polypeptide bands were excised from the dried gel and assayed for radioactivity by liquid scintillation counting.

UV TRANSCRIPTION

UNIT

MAPPING

IN Ad 2

79

FIG. 2. SDS-polyacrylamide-gel autoradiogram of labeled extracts of AD a-infected whole cells after various doses of uv irradiation at 10 hr postinfection, and of purified virus. The cell extracts were labeled for 1 hr with [Wmethionine at 16 hr postinfection. Adenovirus was purified as described in the text. (A) 0, (B) 50, (C) 100, (D) 150, (FJ ZOO, and (F) 250 ergs/mm2; (G) mock-infected KB cells; (H) purified adenovirus.

Ad 2 proteins. Employing the autoradiogram as a guide, protein bands were excised from the dried gel and their radioactivity was assayed by liquid scintillation counting. Typical radioactivity counts per minute obtained from such a gel are shown in Table 1. From the radioactivity obtained in each polypeptide band, inactivation curves for the synthesis of the various late Ad 2 proteins were determined. The average of the results from two polyacryl-

amide gels of the same experiment are shown in Figs. 3 and 4. A comparison of the uv-inactivation curves for the genes IIIa and IV demonstrates that the uv sensitivity is not merely a function of gene size, since proteins IIIa and IV are of similar molecular weight (Anderson et al., 19731, but yield very different inactivation profiles. Furthermore, it can be seen that uv irradiation resulted in an increase in the amount

80

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AND

detected for one of the proteins, pV1, the precursor to protein VI, as compared to a decrease for all other late viral proteins. This is thought to result from a radiation effect on the processing of pV1 occurring during the 1-hr labeling period. Both pV1 and pVI1 are processed following their synthesis (Anderson et al., 1973), and as a consequence the amount of labeled protein detected in the corresponding polypeptide bands may not truly represent the amount of these proteins actually synthesized during the labeling period. Lewis and coworkers (1975) have found that the genes for polypeptides II (hexon), IIIa, III (penton), V (minor core), pV1, and pVI1 (major core precursor) are located on the endonuclease EcoRI-A fragment, which encompasses about 60% on the left-hand end of the viral genome, whereas genes 1OOK and IV (fiber) have been assigned to the right 30% of the viral genome. By means of hybridization techniques and cell-free translation these genes representing the majority of late Ad 2 proteins have been assigned to the light strand of the viral DNA (Oberg, Mak, and Philipson, personal communication). This assignment of groups of late genes to particular regions of the Ad 2 genome, together with the uvinactivation curves reported here indicate the gene order within each of two independent transcription units on the light strand for the majority of the late Ad 2 proteins. Transcription from a single promotor

RAINBOW

would result in a significantly greater inactivation of protein synthesis for genes 1OOKand IV as compared to gene II, since genes 1OOKand IV are both distal to gene II. However, it can be seen from Figs. 3 and 4 that the inactivation of polypeptide synthesis for genes 1OOKand IV is in fact far less than that for gene II, indicating the existence of a second transcriptional unit encoding genes 1OOK and IV. The inactivation curves shown in Figs. 3 and 4 indicate that the first transcription unit has the gene order IIIa, V, III, and II, whereas the second unit consists of gene 100K followed by gene IV. Due to the processing of proteins pV1 and pVI1 the relative positions of these genes encoding these proteins could not be determined directly from the present study. It is seen from Figs. 3 and 4 that, with the exception of gene pV1, increasing uv doses resulted in an exponential decrease for each gene product synthesized. The exponential slope of each uv-inactivation curve reflects the uv sensitivity of the genes coding for the various late Ad 2 proteins. K, the slope of each inactivation curve, is assumed to be proportional to the length along the DNA from the promotor to the 5’-terminus of each gene as has been found in other systems (Sauerbier et al., 1970; Ball and White, 1976). Data points from duplicate gels of the same experiment were fitted by leastsquares analysis to obtain the K values shown in Table 2. The standard error of

TABLE [?~]METHIONINE Dose (ergs/ mm*) 0* 50 100 150 200 250

IIIa

1OOK

10,300 10,000 9,800 8,800 8,500 7,500 ND’

28,800 32,000 26,900 27,300 21,500 20,800 18.500

1

COUNTS PER MINUTE IN LATE Ad 2 PROTEIN BANDER II III pv11 IV V

15,300 14,200 14,400 12,900 12,100 9,000 ND

12,300 11,600 10,100 8,900 8,600 6,500 5.500

9,100 8,100 8,000 6,600 4,800 4,800 3.700

15,700 18,900 14,900 12,200 12,700 5,200 8.900

62,900 53,400 44,400 41,400 29,800 30,200 12.700

PVI

5,100 4,900 5,300 5,200 5,100 5,000 ND

a Background counts per minute were obtained by assaying gel fragments from regions of the gel where no discrete bands were detected on the corresponding autoradiogram. An average background of 1200 cpm has been accounted for in the above data. * A repeat at 0 ergs/mm2 was run in parallel as a measure of the variability inherent in the procedure. e Not done.

UV TRANSCRIPTION

UNIT

\ A

uv

~0s~

200 (ERGS

81

IN Ad 2

fragments (Lewis et al ., 1975; Atkins et al., 1975), the determination of the location for the antigenic determinants of the hexon (gene product II) and fiber (gene product IV) on the Ad 2 genome (Mautner et al., 19751, and the relative gene order determined by the study reported here. It can be seen that a plot of the K value for each gene within a single transcription unit versus its physical distance along the Ad 2 DNA yields a linear curve as expected. The intercept on the abscissa thus represents the physical location on the adenovirus genome for the initiation of

q

100

MAPPING

31 MM-~)

FIG. 3. Relative rates of synthesis of late Ad 2 proteins from infected whole cells, as a function of uv dose, for the proteins known to be coded for by the first 60% of the Ad 2 genome (Lewis et al., 1975). The points of the graph were obtained from evaluating autoradiograms such as in Fig. 2. Each point is the average of results from two separate SDS-polyacrylamide gels of the same experiment. The abscissa gives the uv dose to the infected cell monolayers at 10 hr postinfection; the ordinate displays the normalized rate of synthesis of several late proteins. Closed squares, gene pV1; open circles, gene IIIa; open triangles, gene V; closed circles, gene III; open squares, gene pVI1; closed triangles, gene II.

uv

the slopes shown gives an indication of the error involved in this type of experiment. In Fig. 5, the inactivation cross section, K, for each of the different late proteins is plotted against a proposed position on each transcription unit. The proposed positions of the Ad 2 late genes considered have been assigned on the basis of the molecular weights of Ad 2 late gene products (Anderson et al., 1973), the assignment of late polypeptides to restriction enzyme

DosE

(ERGS rwuI-3

FIG. 4. Relative rates of synthesis of late Ad 2 proteins from infected whole cells as a function of uv dose for proteins known to be coded for by the right 30% of the Ad 2 genome (Lewis et al., 1975). The points on the graph were obtained from evaluating autoradiograms such as in Fig. 2. Each point is the average of results from two separate SDSpolyacrylamide gels of the same experiment. The abscissa gives the uv dose to the infected cell monolayers at 10 hr postinfection; the ordinate displays the normalized rate of synthesis of several late proteins. Open circles, gene 100K; closed squares, gene IV.

82

GIRVITZ

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-I,:r?

RAINBOW

tm:4%cj-Jj

GENE

t

GENE IO

5

IS

t

1 5

lb

I5

NO. OF BASE PAIRS (xIO-~) FIG. 5. Ultraviolet sensitivity of the production of Ad 2 late proteins versus the proposed physical position of late Ad 2 genes for each transcription unit. The proposed physical position is based on the molecular weights of Ad 2 late gene products (Anderson et al., 1973), the assignment of late polypeptides to restriction enzyme fragments (Lewis et al., 1975; Atkins et al., 1975), the determination of the location for the antigenic determinants of the hexon (gene product II) and fiber (gene product IV) in the Ad 2 genome (Mautner et al., 1975), and the relative gene order determined by the study reported here. An area corresponding to the molecular weight of gene pV1 was accounted for preceding gene II (not shown). The abscissa gives the late region of the Ad 2 genome in base pairs and the position of late genes; the ordinate reflects the uv sensitivity of late genes’of Ad 2. The K value is the slope for the inactivation curve of each gene product in Figs. 3 and 4 calculated from the equation, K = (In P,/P)/D, where PO = relative rate of protein synthesis at 0 uv dose and P = relative rate of protein synthesis at uv dose D. TABLE

2

ULTRAVIOLET SENSITIVITY OF THE LATE GENES OF Ad 2 Gene

K (x 10e3) (mmz/erg)O

Relative

radiosensitivity*

Expt 1 IIIa V III pv11 II 1OOK IV

1.58 2.15 2.83 3.18 4.62 1.87 3.92

? 2 k ? c 2 A

0.18 0.50 0.18 0.39 0.51 0.38 0.80

0.34 0.47 0.61 0.69 1.00 0.41 0.85

+ + + + k r +

0.07 0.15 0.11 0.16 0.22 0.12 0.36

Expt 2’ 0.22 k 0.03 0.34 k 0.04 0.54 * 0.05 NDd 1.00 + 0.09 0.40 -t 0.05 0.78 + 0.07

,J Data points from duplicate gels of Expt. 1 were fitted by least-squares analysis to obtain the standard errors shown. b Relative radiosensitivity as compared to gene II. c Cells were uv irradiated 9 hr postinfection and pulse labeled at 15 hr for 0.5 hr. The relative radiosensitivity of each protein was determined from the slab-gel autoradiograms by microdensitometry. d Not done.

each transcript. The slopes of the curves in Fig. 5 are characteristic of the sensitivity of the RNA polymerase to the termination of transcription by the uv-induced

lesion in the viral DNA. The similarity in slopes for the two transcriptional units shown in Fig. 5 suggests that both transcriptional units are utilizing the same polymerase and that the uv transcriptionterminating lesions are produced tbroughout the DNA segments with approximately equal frequency. This experiment has been repeated several times with slight variations in the time of uv irradiation postinfection as well as in the time of pulse labeling. In one experiment Ad 2-infected KB cells were uv irradiated at 9 hr postinfection and pulse labeled for 0.5 hr at 15 hr postinfection. The relative radiosensitivity of each protein was then determined from the slab-gel autoradiograms by microdensitometry. The relative radiosensitivities obtained for each of the late Ad 2 genes in this experiment are shown in Table 2. In this and all experiments, the radiosensitivity of genes 1OOK and IV was found to be less than that for gene II, indicating the existence of two independent transcriptional units on the light strand of Ad 2. Furthermore, the order of radiosensitivity for the genes within each

UV TRANSCRIPTION

UNIT

transcriptional unit was the same for all experiments indicating the relative gene order for the late Ad 2 genes as described. DISCUSSION

When Ad 2-infected cells are exposed to increasing doses of uv irradiation at 10 hr postinfection, the amount of each specific late gene product synthesized was found to be differentially reduced. This differential uv inactivation of late viral proteins is thought to result from the induction of lesions on the viral DNA template that cause the premature termination of RNA chains (Sauerbier, 1975). The probability that a given gene will become expressed then becomes a function of the effective target size on the viral DNA, i.e., the physical distance between the 5’-terminus of that gene and the promotor for that transcript. Inactivation curves for the synthesis of each of the late viral protein were examined on this basis. Using a preliminary map determined by the assignment of genes for late polypeptides to restriction enzyme fragments (Lewis et al., 1975), together with the assignment of the majority of these genes

MAPPING

IN Ad 2

83

to the light strand of the Ad 2 genome (Oberg, Mak, and Philipson, personal communication), the results presented in this work suggest that there are two transcription units on the light strand on the Ad 2 genome which code for the majority of the late Ad 2 proteins. Furthermore, we have been able to order some of the genes within each transcription unit. We propose that the first transcriptional unit includes the genes IIIa, V, III, pVI1, and II in that order, and that the second unit consists of gene 1OOKfollowed by IV also in that order as shown in Fig. 5. Current data are consistent with the map shown in Fig. 6, although the precise locations on the genome for each late gene examined could not be determined from this work. The positioning of gene pVI proximal to gene II, as shown in Fig. 6, is based on the finding of Lewis et al. (1975) that gene pVI is linked to gene II at the boundary of the EcoRI-A and -B fragments and on the position of gene II as described previously in this report. However these data do not exclude the possibility that gene pV1 is distal to gene II. The data obtained in Fig. 3 are incon-

3-I 5’h

; ECORI

FRACTlON OF AD2 GENOME LENGTH FIG. 6. A putative map for the light strand of the Ad 2 genome. The positions of cleavage by the restriction enzyme EcoRI were determined by Mulder et al. (1974). The transcriptional maps of early and late RNA have been determined by Flint et al. (1975b) and Pettersson et al. (1976). The nomenclature for late polypeptides follows the designation according to Anderson et al. (19731, Eve&t et al. (1973), and Lewis et al. (1974). The positions of early gene products are based on the fragment assignment of Lewis et al. (1976). The assignment of groups of late genes to particular regions of the Ad 2 genome was determined by Lewis et al. (1975) and Atkins et al. (1975). Specific gene order within the two transcription units for late Ad 2 proteins was obtained from this work. The region designated NER (nonessential region) is probably not required for Ad 2 replication (Flint et al., 1975a; Pettersson et al., 1976) and corresponds to the position and extent of the deletion of adenovirus DNA in Ad 2 +ND, (Kelly and Lewis, 1973). PR denotes the proposed position for the promotor sites for the transcription of each of the late transcriptional units. Dark and light blocks designate the late and early regions of transcription, respectively. The thin line separated by the small vertical bars delineates each gene.

84

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sistent with the positioning of gene pVI on the map in Fig. 6. This inconsistency may be explained by the fact that pVI, the precursor to protein VI, may be synthesized and then processed during the lh pulse period resulting in the detected amounts of pVI not being representative of the true quantity of pVI synthesized during that period. The increase in the amount of labeled pVI detected after irradiation may result from uv-induced reduction of processing for pVI. The existence of processing may not allow the mapping of precursor polypeptides by this technique. The processing step for precursor polypeptides also places doubt on our positioning of gene pVI1. Another viral-coded protein is pVII1, a precursor polypeptide to one of the proteins of the virion. The position for pVII1 is anomolous as it has been found to map on two distinct EcoRI restriction fragments of the Ad 2 genome. This is most likely due to a second polypeptide with the same apparent molecular weight comigrating on polyacrylamide gels with pVII1 (Lewis et al., 1975). Consequently, pVII1 was not amenable to uv transcriptional unit mapping. The position for gene II (hexon) and gene IV (fiber) is consistent with a previous report determining the location for the type-specific antigenic determinants by a serological analysis of these capsid antigens from 17 Ad 5-Ad 2 +NDl recombinants (Mautner et al., 1975). Several recent reports (Bachenheimer and Darnell, 1975; Goldberg et al., 1977; Weber et al., 1977; Vennstrom and Philipson, 1977) provide data to suggest that a large proportion of nuclear RNA is transcribed from adenovirus DNA as relatively long RNA molecules. However, some of these studies and another report (R. Weinmann, Wistar Institute, personal communication) have also suggested the possibility of the existence of more than one promotor for the synthesis of late viral RNA. The data of this report suggest that there are at least three promotors for messenger RNA on the light strand of Ad 2: one at the origin of transcription for early RNA at the extreme left-hand end (Pettersson et al., 1976) and two other promotors, one

RAINBOW

for each of the late transcripts described in this report (Fig. 6). Since only 10 to 20% of the total nuclear viral RNA is transported to the cytoplasm late in adenovirus infection (Philipson et al., 1974),, the majority of the relatively long nuclear viral RNA molecules described by several workers (Bachenheimer and Darnell, 1975; Goldberg et al., 1977; Weber et al., 1977; Vennstrom and Philipson, 1977) are not precursors of cytoplasmic messenger RNA. The results of this report suggest that these relatively long nuclear viral RNA molecules are not precursors of cytoplasmic messenger RNA for the synthesis of proteins 1OOKand IV. It has been suggested that the majority of uv-induced transcription-terminating lesions on the DNA are pyrimidine dimers (Sauerbier, 1975). Assuming this and using the data of Rainbow and Mak (1973) for the rate of thymine dimer induction in Ad 2 DNA, i.e., 7.5 x lop3 dimers/single strand/erg/mm2, it can be calculated that one lethal hit for each transcriptional unit corresponds to about 0.7 thymine dimer. Thus, assuming that the thymine dimers are transcription-terminating lesions, they can account for at most 70% of the transcription-terminating lesions, leaving the other 30% unknown. This is consistent with studies on bacterial DNAs in which only 70% of the uv-induced damage is able to be photoreactivated (Swenson, 1972). However, it is also possible that the value of 0.7 thymine dimer per transcriptionterminating lesion for Ad 2 reflects, in part at least, repair of thymine dimers in the 6 hr between the uv irradiation and the pulse label. ACKNOWLEDGMENTS We thank Dr. Frank Graham, Mr. Norman Lassam, Dr. Stanley Mak, and Dr. Jim Smiley for their help during the course of this work. This investigation was supported by the National Cancer Institute of Canada. REFERENCES ANDERSDN, C. W., and GESTELAND, R. F. (1972). Pattern of protein synthesis in monkey cells infected by simian virus 40. J. Viral. 9, 758-765. ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1973). Processing of adenovirus a-induced pro-

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Ultraviolet transcriptional unit mapping for the late genes in adenovirus type 2.

VIROLOGY 84, 75-86 (1978) Ultraviolet Transcriptional Unit Mapping for the Late Genes in Adenovirus Type 2 SHELDON Departments of Biology C. GIR...
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