Characterization of transcription-deficient temperature-sensitive mutants of herpes simplex virus type 1

VIROLOGY

91, 364-379 (1978)

Characterization of Transcription-Deficient Temperature-Sensitive Mutants of Herpes Simplex Virus Type 1 ROGER J. WATSON’

AND J. BARKLIE

CLEMENTS’

Institute of Virology, University of Glasgow, Church Street, Glasgow GlI 5JR, Scotland Accepted August 18, 1978 Six DNA-negative, temperature-sensitive (ts) mutants (ts B, ts D, ts E, ts K, ts S, and ts T) of herpes simplex virus type 1, which fall into four complementation groups, have been characterized by analyses of the nuclear and cytoplasmic transcripts synthesized in cells infected at the restrictive temperature. Transcripts were hybridized to blot strips containing the separated fragments of virus DNA generated by digestion with restriction endonucleases Hind III, Hpa I, or Barn Hl. The hybridization patterns thus obtained have been compared with the patterns given by RNA isolated from cells infected with wild-type virus and labeled at early and late times postinfection (before and after viral DNA replication), or in the continuous presence of inhibitors of DNA or protein synthesis (Clementa, J. B., Watson, R. J., and Wilkie, N. M. (1977). Cell 12,275-285). Of these mutants, ts K is the most restricted in transcription and gives a pattern of hybridization similar to that observed with immediateearly RNA (transcribed in the absence of protein synthesis). Two mutants (ts D and ts T), which lie in a different complementation group to ts K, also give restricted hybridization patterns but express additional regions of the genome as compared with ts K RNA. Inasmuch as these three mutants fall into two complementation groups, these experiments suggest that at least two viral products are required to progress from the immediate-early to the early stage of transcription. The hybridization patterns obtained with the other early mutants examined here (ts B, ts E, and ts S) are, in contrast, much less restricted, and resemble the early, rather than late pattern. Downshift of ts K-infected cells from the nonpermissive to the permissive temperature, both in the presence and absence of further protein synthesis, results in the transcription of regions which map throughout the viral genome. These experiments suggest, therefore, that the polypeptides which are required for the switch-on of these additional transcripts accumulate in ts K-infected cells under restrictive conditions. INTRODUCTION

inhibitors of DNA or protein synthesis. Cytoplasmic RNA synthesized in the continThe temporal regulation of the transcrip(presumtion of herpes simplex virus type 1 (HSV-1) uous presence of cycloheximide has been examined previously both by ably transcribed by pre-existing cellular enRNA excess hybridization, in liquid phase, zymes and referred to here as immediateand by the blot hybridization procedure of early (IE) RNA) has been reported to be homologous to only 10% of the viral DNA Southern (1975). The liquid hybridization technique has (Kozak and Roizman, 1974). In contrast, allowed an estimate of the proportion of RNA extracted from cells at early and late viral DNA represented by transcripts ac- times was complementary to 44 and 47% of the DNA, respectively (Frenkel and Roizcumulating in both nucleus and cytoplasm before and after viral DNA replication (at man, 1972). For late RNA, few sequences early and late times postinfection), and in could be detected in the nucleus which were not present in the cytoplasm (Kozak and celIs treated from the time of infection with Roizman, 1974). The blot hybridization procedure has en’ Medical Research Council research scholar. of the genome location ‘Author to whom reprint requests should be ad- abled determination dressed. of transcripts which accumulate under var0042-6822/78/0912-0364$02.00/O Copyright

0 1978 by Academic

Press, Inc.

All rights of reproductionin any form reserved.

364

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ious labeling regimes. The hybridization of both nuclear and cytoplasmic IE RNAs was restricted to certain regions of the genome only, and these were mostly in, or proximal to, the inverted repeat sequences (Clements et al., 1977). However, both early and late RNA, and RNA labeled in the continuous presence of cytosine arabinoside, appeared qualitatively similar, and hybridized to all the DNA restriction fragments used in the analyses (Clements et al., 1977). Quantitative differences between early and late RNAs, however, were observed. The genome locations of cytoplasmic IE and late RNAs obtained by liquid hybridization (Jones et al., 1977) are in good general agreement with our published data obtained by blot hybridization. These results suggest that at least two major transcriptional controls act upon HSV-1 gene expression. The first is an offon control and requires protein synthesis to switch-on early transcription. The second is an abundance control which is dependent upon viral DNA synthesis for the change in transcript abundance. Analyses of RNA isolated from cells at various times postinfection, and from cells treated with inhibitors of protein or DNA synthesis cannot, per se, provide information on the effect of specific virus gene products on the regulation of transcription. The use of temperature-sensitive (ts) mutants of the virus allows such information to be obtained. For example, it has been shown using ts mutants that at least two viral products are required for the switch from early to late transcription with adenovirus type 5 (Berget et al., 1976; Carter and Ginsberg, 1976). A number of ts mutants of HSV-1 have been identified and complemented (Brown et al., 1973; Crombie, 1975), and the polypeptides synthesized in cells infected with these mutants at the permissive (PT) and nonpermissive temperatures (NPT) have been characterized (Marsden et al., 1976). These mutants can be placed into two major classes,those which replicate their DNA at the NPT (DNA +ve) and those which do not (DNA -ve). The DNA -ve ts mutants have been found, in general, to be more defective than DNA +ve ts mutants

ts MUTANTS

365

OF HSV-1

in terms of the numbers of virus-specific proteins synthesized (Marsden et al., 1976) and virus-specific enzyme activities detectable at the NPT (Subak-Sharpe et al., 1974; Crombie, 1975). Here, we report on the use of the blot hybridization procedure to map transcripts synthesized in BHK Cl3 cells infected with six DNA -ve ts mutants of HSV-1 strain 17. The six ts mutants examined here (ts K, ts D, ts T, ts B, ts E, and ts S) fall into four complementation groups because ts D and ts T, as well as ts B and ts E, fail to complement (Brown et al., 1973; Crombie, 1975). Of these mutants, ts K is the most restricted and makes very few virus-specific polypeptides at the NPT, whereas the other mutants synthesize many but not all of the wild-type polypeptides. MATERIALS

AND

METHODS

Cells. BHK 21 (C13) cells were grown as monolayers in 90-mm Petri dishes in Eagle’s medium supplemented with 10%tryptose phosphate and 10% calf serum. Near confluent monolayers (approximately 1.5 x 10’ cells in total) were incubated for 16 hr in low-phosphate Eagle’s medium containing 2% calf serum prior to virus infection. Virus. The temperature-sensitive (ts) mutants used in this study (ts B, ts D, ts E, ts K, ts S, and ts T) were derived from the wild-type (wt) of HSV-1 (Glasgow strain 17) as described by Brown et al. (1973) and Crombie (1975). Ts B, ts D, and ts S had the syn+ plaque morphology, whereas ts E, ts K, and ts T were of the syn plaque morphology. Ts mutant stocks were grown at the permissive temperature (31”) and titrated at both the permissive and nonpermissive (38.5“) temperatures to check for reversion. Titers at the permissive temperature (PT) were at least 104-fold greater than those obtained at the nonpermissive temperature (NPT). The revertant of ts D syn+, ts D/R4, was isolated by picking a plaque which arose spontaneously at NPT and was subjected to several rounds of plaque purification (Taylor, 1976). RNA labeling in wt and ts mutantin-

366

WATSON AND CLEMENT.9

fected cells. Cells infected with the wt were

labeled with [32P]orthophosphate early (O-l hr postinfection), late (O-7 hr postinfection), and in the presence of metabolic inhibitors, as described previously (Clements et al., 1977). Cells were infected with ts mutants at a multiplicity of 50 PFU/cell either under restrictive or permissive conditions. Under restrictive conditions, the virus was allowed to adsorb for 1 hr at NPT. After adsorbtion, virus was removed and monolayers were washed with low-phosphate Eagle’s medium pre-warmed to 38.5”. Pre-warmed, low-phosphate Eagle’s medium (5 ml) containing 2% calf serum and 0.5-l mCi/ml [“‘Plorthophosphate was then added to each monolayer, and the cells were incubated for 7 hr at NPT. Under permissive conditions, the virus was adsorbed, washed, and labeled at PT otherwise using the same protocol as above. The amount of HSV messenger RNA, as determined by in vitro translation (C. M. Preston, personal communication), and also the virus yield were unaffected by incubation in low-phosphate medium for the times used in these experiments. Cell fractionation

and RNA extraction.

After labeling, monolayers were washed with cold isotonic saline, and the cells scraped off and pelleted. Nuclear and cytoplasmic cell fractions were prepared as described by Zieve and Penman (1976). Cytoplasmic cell fractions (6 ml) were made 0.01 M with respect to EDTA, 0.5% with respect to SDS, and proteinase K (Boehringer Mannheim, GmbH) was added to 500 pg/ml. Pelleted nuclei were resuspended in PK buffer (0.01 M EDTA, 0.01 M NaCl, 0.01 M Tris-HCl, pH 8.0), made 0.5% with respect to SDS and 500 pg/ml proteinase K were added. Cell fractions were incubated at 22” for 16 hr, and were extracted once with phenol and once with chloroform containing 2% isoamyl alcohol. Nucleic acids were precipitated by the addition of 3 volumes of ethanol. After storage overnight at -2O”, nucleic acids were pelleted by low-speed centrifugation, washed with ethanol, and then were dissolved in 1 ml DNase buffer (0.1 M CH&OONa, 2.5 m&f Mg,SO?, pH 5.0). Electrophoretically

purified DNase 1 (Worthington Bidchemical Corp.) was added to 25 pg/ml, and incubation was for 30 min at 37”. The samples were applied to a Sephadex G-50 column (1.5 x 25 cm) and eluted with NETS buffer (0.1 M NaCl, 0.01 M EDTA, 0.05 M Tris-HCl, pH 7.5, 0.1% SDS). Fractions comprising the initial peak of radioactivity eluted were combined, and the RNA was precipitated with ethanol. The final RNA purification procedure was by isopycnic banding in cesium sulfate. Pelleted RNA samples were dissolved in 0.1 x SSC (1 x SSC is 0.15 M NaCl, 0.015M sodium citrate, pH 7.5) and C&SO? was added to give a final density of 1.60 g/ml. Samples were centrifuged at 50,000 r.p.m. in a MSE 8 x 14 fixed-angle rotor for 60 hr at 4’. Under these conditions, single-stranded RNA forms a sharp band (SzybaIski, 1968) which allows the complete separation of RNA from any remaining DNA. Unless the RNA preparations were preannealed extensively prior to banding, no RNA banding at a density characteristic of double-stranded RNA was observed. Nick translation of HSV-1 DNA. HSV-1 DNA was labeled to high specific activity (10’ cpm/pg) by nick translation with DNA polymerase 1 (Boehringer Mannheim GmbH) and all four cc-32P-deoxyribonucleoside triphosphates (specific activity, 80-250 Ci/mmol, obtained from the Radiochemical Centre, Amersham, U.K.) using the procedure of Rigby et al. (1977) essentially as described by Wilkie (1976). Endonuclease digestions. Reaction mixtures contained, in a volume of 400 ~1: 10 r&f Tris-HCl, pH 7.9, 6 n-J4 MgCl?, 6 n-&! ,L?-mercaptoethanol, 0.02% bovine serum albumin, lo-18 pg HSV-1 DNA, and sufficient endonuclease to produce a limit digest. Reactions were terminated after 4 hr at 37” by addition of 100 ~1 of 5 x electrophoresis buffer (1 x electrophoresis buffer is 0.036 M Tris, 0.03 M NaH?PO+ 0.01 M EDTA, pH 7.8, containing 50% sucrose and 0.2% bromphenol blue. Agarose gel electrophoresis. Hind III, Hpa I, and Barn Hl DNA fragments were separated, respectively, on 0.4,0.5, and 0.8% agarose gels, essentially as described previously (Clements et al., 1976).

TRANSCRIPTION-DEFICIENT

Blot hybridization procedures. The denatured DNA fragments were transferred from the agarose gels onto nitrocellulose membranes using the procedure of Southern (1975). Strips (approximately 5 mm wide) were cut lengthwise from the nitrocellulose sheet such that each blot strip contained all the fragments of unlabeled DNA (0.5-l pg total DNA per strip). Hybridization, autoradiography, and densitometer tracings were performed as described previously (Clements et al., 1977). RESULTS

Transcripts in ts K-, ts D-, and ts T-infected cells Analysis of the mapping data requires a knowledge of the HSV-1 DNA structure. The genome has an unusual structure (Sheldrick and Berthelot, 1974; and Fig. 9), containing two unique regions (UL and US) each flanked by inverted repeated regions (TRL/IRL, TRs/IRs). A prediction of this configuration is that the unique regions may be inverted relative to each other, either by recombination or replication events, thus generating four possible genome arrangements (Hayward et al., 1975; Clements et al., 1976). A further consequence of the HSV-1 DNA structure is that fragments produced by restriction enzymes may contain sequences which are present in other fragments generated by the same digest (Wil kie and Cortini, 1976). Hybridization, therefore, of RNA to a fragment containing any part of the inverted repeat regions does not necessarily imply that this RNA was transcribed from that region of the genome, but merely that it is complementary to part of that fragment. Physical maps for the HSV-1 DNA fragments generated by digestion with several restriction endonucleases have been obtained (Wilkie, 1976; A. Davison and N. M. Wilkie, personal communication). The Hpa I, Hind III, and Barn Hl cleavage maps used here are shown in Fig. 8. Hybridization of [““PIRNA to blot strips containing the denatured fragments of HSV-1 DNA generated by digestion with various restriction endonucleases, and subsequent visualization of bound RNA by autoradiography,

ts MUTANTS

OF HSV-1

367

allows the map location of viral DNA complementary to the labeled transcripts to be determined. Cells infected at the NPT with ts K, ts D, or ts T were labeled with [32P]orthophosphate from O-7 hr postinfection. Cytoplasmic and nuclear RNAs were isolated and hybridized to blot strips containing the denatured fragments of HSV-1 DNA generated by digestion with Hpa 1, Hind III, or Barn Hl restriction endonucleases (Figs. 1-3). For comparison, the hybridization patterns of IE RNA, from cells infected with wt HSV-1 in the continuous presence of cycloheximide, are also shown (Figs. l-3). In addition, RNA labeled in cells infected at the NPT with a spontaneous revertant of ts D, ts D/R4 (Taylor, 1976), is shown hybridized to Hpa 1 fragments (Fig. l), and RNAs labeled in cells infected with ts K, ts T, and ts D at the PT are shown hybridized to the Hind III digest (Fig. 2). IE RNA, and RNA from cells infected at the NPT with ts K, ts D, and ts T, contain an abundance of material which hybridizes to Hpa 1 a, cde, gh, m, and s (Fig. 1). In addition, there is readily detectable hybridization to f and 1. Fragments a, c, d, g, and m each contain either one or both of the inverted repeats, while s, f, and 1map within Ui. (Fig. 8). IE RNA, ts D RNA, and ts T RNA, but not ts K RNA, show additional hybridization to n and b and, at lower levels, to the remaining Hpa 1 fragments. The pattern of hybridization obtained with ts D/R4 RNA is very different from these restricted patterns and resembles that of wt (Clements et al., 1977). The Hind III digest (Fig. 2) provides similar evidence to that obtained with Hpa 1. IE RNA, and ts K, ts T, and ts D RNAs labeled at the NPT all hybridize to m, which contains TRs (Fig. 8). In contrast to these other RNAs, ts K RNA shows no detectable hybridization to j and little to 1. Cytoplasmic RNAs isolated from cells infected at the PT with these mutants give the wt hybridization pattern (Fig. 2). At the PT, in cells infected with all these mutants, virus-induced polypeptide synthesis is qualitatively and quantitatively identical to that of wt (Marsden et al., 1976). Restriction endonuclease Barn Hl intro-

- -

-

.- - --

- -

- - -- -

-

--

--

-

- _- -- -

..-

, - ^

..-

. - . -

._

._ .

FIG. 1. Autoradiographs of cytoplasmic (C) and nuclear (N) RNA samples hybridized to the Hpa 1 fragments of HSV-1 DNA. RNA was labeled from O-7 hr postinfection at the NPT in cells infected with the mutants ts 1’. ts K, ts D, and ts D/R4 (a spontaneous revertant of ts D), and in cells infected with wt HSV-1 in the continuous presence of cycloheximide (IX). An autoradiograph obtained by hybridizing nick-translated HSV-1 DNA also is shown. *indicates a partial digestion product present on these blot strips.

_

TRANSCRIPTION-DEFICIENT

ts MUTANTS

OF HSV-I

369

FIG. 2. Autoradiographs of cytoplasmic RNA samples hybridized to the Hind III fragments of HSV-1 DNA. Cells infected with ts T, ts K, and ts D were labeled O-7 hr postinfection at either the NPT or the PT. Cells infected with wt were labeled from O-7 hr postinfection (late), and in the continuous presence of cycloheximide GE).

duces many more cuts into HSV-1 DNA than either Hpa 1 or Hind III, and the cleavage map (Fig. 8) has allowed finer mapping of transcripts. Figure 3 shows IE RNA, ts K, ts T, and ts D RNAs hybridized to the Barn Hl fragments. There is, in all

these RNAs, an abundance of material which hybridizes to b, e, n, x, and z. Fragments b and e contain the same TRL/IRL sequences, but have different sequences from UL, while n, x, and z map in the short region. Compared with the hybridization

370

WATSON

AND

CLEMENT.9

FIG. 3. Autoradiographs of cytoplasmic (C) and nuclear (N) RNA samples hybridized to the Barn Hl fragments of HSV-1 DNA. RNA was labeled from O-7 hr postinfection in cells infected with the ts mutants (ts T, ts K, ts D or ts E) or with wt in the continuous presence of cycloheximide (IE).

patterns of IE and ts K RNAs, ts D and ts T RNAs show additional hybridization to m, r, uv, and w. There is little evidence for qualitative differences of transcripts between nucleus and cytoplasm. However, with IE RNA there is readily detectable hybridization of nuclear RNA to Hpa 1 opqr, but little with cytoplasmic RNA (Fig. 1). For ts T and ts D, but not ts K, RNAs there is some indication of greater relative hybridization to certain fragments with cytoplasmic RNA

samples, for example, to Hpa 1 f (Fig. l), than with nuclear RNA samples. Transcripts Cells

in ts B-, ts E-, and ts S-infected

Nuclear and cytoplasmic RNA from cells infected and labeled at the NPT with ts B, ts E, or ts S are shown hybridized to the Hpa 1 fragments (Fig. 4). For comparison, the hybridization patterns obtained with cytoplasmic RNA isolated from cells infected with wt HSV-1 and labeled at early

TRANSCRIPTION-DEFICIENT

ts MUTANTS

OF HSV-1

371

c,d

FIG. 4. Autoradiographs of cytoplasmic (C) and nuclear (N) RNA samples hybridized to the Hpa 1 fragments of HSV-1 DNA. Cells infected with ts B, ts S, and ts E were labeled from O-7 hr postinfection at NPT. RNA was isolated from wt infected cells labeled from O-l hr postinfection (early), or from O-7 hr postinfection in the absence (late) or presence of ara C.

and late times, and also in the continuous presence of cytosine arabinoside (ara C), are shown. For all these RNA samples, there is hybridization to every DNA fragment generated by this enzyme digest, but there appear to be quantitative differences between certain of these hybridization patterns. Because hybridizations were performed in at least lo-fold excess of DNA over HSV-1

RNA, the relative amount of RNA homologous to a particular DNA fragment should be directly proportional to the intensity of the autoradiographic image. Therefore, autoradiographs were scanned with a microdensitometer to estimate the relative amount of hybridization to each DNA fragment, and these are shown for several cytoplasmic ,RNAs (Fig. 6). Early and late RNA, ts B, ts E, and ts S

372

FIG. infected isolated absence

WATSON

AND

CLEMENTS

5. Autoradiographs of cytoplasmic RNA samples hybridized to the Hind III fragments of HSV-1. Cells with ts B, ts S, and ts E were labeled from O-7 hr postinfection at either the NPT or the PT. RNA was from cells infected with the wt and labeled O-7 hr postinfection either in the presence of ara C or in its (late).

RNAs all show little hybridization to Hpa 1 m (Figs. 4 and 6), a fragment which is represented abundantly by IE RNA (Fig. 1). The patterns obtained with ts B and ts E, which are in the same complementation group, appear to be identical, but they differ somewhat from those obtained with ts S or with ara C.

Figure 5 shows the Hind III hybridization patterns of cytoplasmic RNAs isolated from cells infected with ts B, ts E, and ts S at the NPT and the PT. The hybridization patterns of RNA from cells infected with wt in the presence of ara C and with late RNA are also shown. Densitometer traces of the autoradiographs are given for several

TRANSCRIPTION-DEFICIENT

ts MUTANTS

OF HSV-1

373 e,ds

ts B

ts s n

v

ut vut

tsE

FIG. 6. Densitometer traces from autoradiographs of cytoplasmic fragments. The autoradiographs scanned were those shown in Fig. 4.

RNA samples hybridized

to the

Hpa 1

tsB

b.a ts E

ara C I

FIG. 7. Densitometer traces from autoradiographs of cytoplasmic RNA samples hybridized fragments. The autoradiographs scanned were those shown in Fig. 5.

to the Hind III

374

WATSON

Map

0

AND

CLEMENT?!

Units

Hind III ioh

1 ,

j

II

Hpa

0.5 0.6 0.7 0.8 0.9

0.2 0:3 04

01

I

I

I o=c+m d =g+m

Barn HI se c 0 I

I

mtpugvrw I III II Ill1 df’i’

de’

FIG. 8. Physical maps for the fragments Hpa 1, and Barn HI.

d I 1 II III

I

ho1 II I

!2ji5

Mapping

Ii

Data

The cytoplasmic map locations for ts K, ts D, and ts E obtained by hybridization to the Hpa 1, Hind III, and Barn Hl fragments are summarized in Fig. 9, and are compared with the locations of cytoplasmic IE RNA. The data have been further confirmed by hybridization of these RNAs to the fragments produced by cleavage with Bgl II or EcoRI and also Hpa l/Hind III in double

k.s+q

c’

of HSV-1 DNA generated

of these cytoplasmic RNAs (Fig. 7). All these RNAs show little hybridization to Hind III m, which contains the TRs sequences. RNAs labeled in cells infected with these mutants at the PT give a hybridization pattern identical to that obtained with wt. In contrast to late RNA, ts B, ts E, and ts S RNAs (labeled at the NPT) and RNA labeled in the presence of ara C show little detectable hybridization to Hind III c and o. At the NPT, therefore, these mutants give the early, rather than the late, hybridization pattern. Nuclear and cytoplasmic RNAs from cells infected with ts E at the NPT have been hybridized to the Barn Hl fragments (Fig. 3). There is almost no detectable hybridization to the terminal fragments (s and q), but abundant hybridization to fragments which map in both UL and Us (a, g, j, n, o, and z). Summary of Transcript

g’

f I 11 11 I

by restriction

endonucleases

Hind III,

digest (data not shown). The findings are summarized as follows: undetectable hybridization to a region is not indicated; low levels of hybridization are indicated bv a dotted line; clearly detectable bands are indicated by a single continuous line; regions to which a relative abundance of material hybridizes are indicated by a double continuous line. It is not possible by these analyses to determine whether the whole or only part of the indicated regions are transcribed. Down-shift

of ts K-infected

Cells to the PT

The transcript mapping data suggest that ts K is blocked at the immediate-early stage of the replicative cycle. In addition, the virus-specific polypeptides which accumulate at the NPT in ts K-infected cells appear to be IE polypeptides,3 i.e., those proteins translated from RNA that has accumulated during a cycloheximide block present since the time of infection (Honess and Roizman, 1974). It was of interest to determine whether these polypeptides which accumulate in ts K-infected cells would, on down-shift from the NPT to the PT, allow expression of additional regions of the genome. ID. McDonald, M. Suh, and H. S. Marsden, mitted for publication.

sub-

TRANSCRIPTION-DEFICIENT

ts MUTANTS

Map Units 0

0.1

0.2

0.3

OL

TRL L

0.5

0.6

0:7

0:8

0:9

IR, IR, -+5-

1 TR,

OF HSV-1

375

cloheximide. These additional regions transcribed map throughout the genome. DISCUSSION

More precise location of the IE transcripts has been achieved by use of the Bum Hl DNA fragments (Fig. 9). Low, but Immedmte Early detectable, levels of hybridization are ap..= _____. __ ____ =-parent to a few noncontiguous regions which map in UL and, in this way, the IE RNA pattern differs from that obtained ts K with ts K at NPT. The relative amount of --I IE RNA which hybridizes to these regions 0 varies somewhat, and thus may represent leakage transcription resulting from residual protein synthesis which occurred even ts T at the high concentration of cycloheximide __.. =...;..=..m -= ____________ _..-.. used (200 pg/ml), as opposed to identifying u a real qualitative difference. Transcription in ts K-infected cells may be blocked rig1s D orously at the IE stage, whereas the drug -_________ - __._ ___-__ _______ block does not exert such a stringent re0 striction; alternatively, there may be regions of DNA transcribed in the absence of ts E protein synthesis which are switched-off by _. -= .. proteins made in ts K-infected cells. m Polyadenylated cytoplasmic IE and ts K FIG. 9. Summary of transcript mapping data. MapRNAs, isolated by selection on oligo(dT)ping data for cytoplasmic RNA from cells infected at cellulose columns, give identical hybridizaNPT with ts K, ts T, ts D, and ts E and for cytoplasmic tion patterns to those obtained with RNAs IE RNA are summarized using the following criteria. isolated by banding in C&O4 gradients (a) undetectable hybridization to a region is not indi(data not shown). This indicates that the cated, (b) low levels of hybridization are indicated by restricted hybridization patterns obtained a discontinuous line; (cl clearly detectable hybridizaare not due to loss of RNA sequences durtion to a region is represented by a single continuous line; (d) regions to which a relative abundance of ing purification, and that all the IE tranmaterial hybridizes are indicated by a double continscripts accumulating during the drug block uous line. are generated by normal rather than aberrant transcriptional mechanisms. It is of interest to compare the polypepCells infected with ts K were incubated at the NPT for 3 hr and then either main- tides which accumulate at the NPT in ts Ktained at the NPT or down-shifted to the infected cells with the IE polypeptides. PT (31’) in the presence or absence of 200 Four polypeptides (V,, 175, 136, 110, and pg/ml cycloheximide. The cells were then 63) are synthesized predominantly in vivo incubated with [“‘Plorthophosphate for 5 on release of a continuous cycloheximide hr and RNA labeled under these various block, present since the time of infection, in regimes was extracted and hybridized to the presence of actinomycin D (Wilkie et blot strips containing the Hpa 1 fragments. al., 1978), and these are the major virusAutoradiographs of the blot strips are specific polypeptides which accumulate at shown in Fig. 10. It can be seen that there the NPT in ts K-infected cells.” Cytoplasis transcription of RNA from additional mic RNA from both ts K-infected cells and regions of the genome on down-shift irre- from cycloheximide-treated cells directs the spective of the presence or absence of cy- synthesis of only these same four polypep0.

“L

“5

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CLEMENTS

FIG. 10. Ts K down-shift, Cells infected with ts K were maintained at NPT for 3 hr and then down-shifted to PT (31”) either in the presence (+CH) or absence (-CH) of cycloheximide. Cytoplasmic (C) and nuclear (N) RNAs, labeled for 5 hr after down-shift, were compared to those labeled for O-7 hr at NPT (38.5”) by hybridization to the Hpa 1 fragments. Hybridization was visualized by autoradiography.

tides in a cell-free translation system.4 These studies from our laboratory, together with the transcript mapping data, strongly suggest that ts K is blocked at the IE stage of the replicative cycle. We have shown (Fig. 10) that the proteins which accumulate in ts K-infected cells are sufficient, on down-shift, to allow 4 C. M. Preston, submitted

for publication

which of regions transcription map throughout the genome. Inasmuch as these additional regions are transcribed both in the presence and absence of cycloheximide, this suggests that down-shift restores the activity of a ts function, presumably a regulatory protein. It is not clear whether all these additional transcripts are functional, but cytoplasmic RNA isolated from ts Kinfected cells after down-shift in the pres-

TRANSCRIPTION-DEFICIENT

ence of cycloheximide directs the synthesis of HSV-l-specific thymidine kinase activity in a cell-free translation system.4 This enzyme is made early in productive infection, but is not detected in ts K-infected cells at the NPT (Crombie, 1975). Moreover, neither IE RNA nor RNA from cells infected with ts K and maintained throughout infection at the NPT contain detectable amounts of HSV-1 thymidine kinase mRNA.4 Inasmuch as we have shown here that down-shift of ts K-infected cells results in the synthesis of additional transcripts, it is probable that this functional mRNA arises by de nouo synthesis rather than by processing of pre-existing RNA. Both ts D and ts T, which fall into the same complementation group, synthesize many more proteins at the NPT than does ts K (Marsden et al., 1976). These mutants do accumulate IE polypeptides under restrictive conditions (especially V,, 175 and llO), and may, therefore, be equivalent to the Houston ts B mutants (Courtney and Benyesh-Melnick, 1974; Courtney et al., 1976). Our mapping data suggest that regions of the genome which are represented abundantly by transcripts isolated from cells infected with these mutants correspond, in part, to those regions represented abundantly by IE and ts K RNA. However, additional regions of the genome also are represented abundantly by ts T and ts D RNA, and these map in both UL and Us. Moreover, there is readily detectable hybridization of these RNAs to fragments which are not represented by cytoplasmic IE RNA (i.e., Bum Hl m and p). Therefore, although IE transcripts and polypeptides accumulate in ts T- and ts D-infected cells, there is also transcription from additional regions of the viral genome and synthesis of some later proteins as compared with ts K-infected cells. The polypeptides specified at the NPT by the revertant of ts D examined here (ts D/R4) have been shown to be qualitatively identical, and quantitatively almost identical, to those specified by the wt (Taylor, 1976). Consonant with this finding, the hybridization patterns obtained with RNA from cells infected with ts D/R4, and labeled until late postinfection, appear iden-

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tical to the wt late hybridization pattern (Clements et al., 1977). The synthesis of IE (or =) polypeptides is shut off during the course of infection, presumably as the result of j3 or y-polypeptide activity (Honess and Roizman, 1974), and this is not prevented by inhibition of viral DNA replication. This shut-off is, therefore, an early function; hence, accumulation of IE polypeptides in ts mutantinfected cells suggests that they are blocked between the IE and early stages of the replicative cycle. The other early ts mutants of HSV-1 examined here (ts B, ts E, and ts S) do not accumulate IE polypeptides at the NPT, but they are defective in the synthesis of a number of virus-specific polypeptides (Marsden et al., 1976). Transcripts synthesized in cells infected with ts B, ts E, or ts S suggest an early, rather than late, program of transcription. Certain regions which are represented abundantly by IE transcripts are scarcely represented in ts B, ts E, and ts S RNA (Fig. 9)) and some regions to which an abundance of ts T RNA hybridizes also are represented abundantly in RNA synthesized by these early mutants. Our data show that ts mutants (ts K and ts T or ts D) which fall into two complementation groups are blocked between the IE and early stages of transcription, and suggest that at least two viral products are required for this progression. It is not clear whether these products switch-on the expression of additional genome regions or are involved in the stabilization or processing of specific transcripts. In cells infected at TNP with ts K for a short period (1 hr), we detected no additional RNA sequences to those shown in Fig. 9. The defective ts K product appears to be an absolute requirement for the expression of early transcripts, whereas additional regions of the HSV-1 genome are transcribed in ts T- (and ts D-) infected cells. It is possible, therefore, that either the defective ts T product may retain some limited function under nonpermissive conditions or, alternatively, this additional transcription is mediated by the regulatory protein which is defective in ts K-infected cells. The physical map locations of a number

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of HSV-1 ts mutants have been determined by marker rescue experiments (Stow et al., 1978). Both ts D and ts K are located in the TRs/IRs region, and it is pertinent that much of the IE RNA hybridizes to this region. Analysis of a number of intertypic recombinants between HSV types 1 and 2 has enabled the genome location of certain polypeptides to be mapped (Wilkie et al., 1978). The V,, 175 IE polypeptide maps in TRs/IRs, and it is, therefore, possible that the lesion of either ts K or ts D is in this polypeptide. Two other IE polypeptides have been assigned genome locations in areas represented by IE transcripts; V,, 110 in TRL/IRL and V,, 63 at the right end of UL between map co-ordinates 0.55-0.81. In addition, a polypeptide of apparent molecular weight 136,000 has been mapped near the middle of UL; it is as yet unclear whether this protein corresponds to the IE polypeptide V,, 136. These studies using ts mutants have allowed us a further insight into the complexity of HSV-1 transcription beyond that obtained using drug blocks. Characterization of further transcription-deficient ts mutants, together with the physical map locations of the lesions and of the polypeptides expressed by these mutants, should lead to a greater understanding of the proteins and processes involved in the regulation of HSV-1 transcription. Note added in proof Recent genetic data (D. Dargan and J. H. Subak-Sharpe, personal communication) indicate that ts K is able to complement both ts T and ts D in the infectious centre complementation test; however positive complementation was not observed in the yield complementation test. Even though ts K recombines well with both ts D and ts T, while ts D and ts T have not been observed to recombine, these new data suggest that ts K, ts D, and ts T cannot unequivocally be assigned to different cistrons.

ACKNOWLEDGMENTS We wish to thank Professor J. H. Subak-Sharpe for his continued interest in this work, and Dr. N. M. Wilkie and A. Davison for providing us with physical maps for the DNA fragments prior to publication. R. J. W. is the recipient of a Medical Research Council grant for training in research techniques. REFERENCES BERGET, S. M., FLINT, S. J., WILLIAMS, J. F., and

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