The Herpes Simplex Virus Type 2 Alkaline DNase Activity Is Essential for Replication and Growth

Transcription

1 J. gen. Virol. (1986), 67, Printed in Great Britain 1173 Key words: HSV-2/DNase activity/dna synthesis~growth The Herpes Simplex Virus Type 2 Alkaline DNase Activity Is Essential for Replication and Growth By HELEN MOSS Institute of Virology, Church Street, Glasgow Gll 5JR, U.K. (Accepted 20 February 1986) SUMMARY A mutant of herpes simplex virus type 2 (HSV-2), which is temperature-sensitive (ts) for the induction of an alkaline DNase activity, was examined at a number of different temperatures. Induction of DNase activity by this mutant resembled that of wild-type (wt) virus at 31 C but was greatly reduced at 38.5 C and barely detectable at 39.2 C. Virus DNA synthesis showed similar patterns, exhibiting wt levels at 31 C, reduced levels at 38.5 C and very little incorporation at 39.2 C. Similarly, virus growth in cells infected with this mutant was equal to that of wt at 31 C, slightly reduced at 38-5 C but considerably reduced at 39.2 C. Marker rescue of the ts DNase lesion restored wt levels of virus DNase activity, of virus DNA synthesis and of virus growth, thus providing direct evidence that HSV DNase activity is essential for virus replication. Herpes simplex virus (HSV)-induced alkaline DNase activity was first reported in HSV-1- infected cells (Keir & Gold, 1963) and later in HSV-2-infected cells (Hay et al., 1971). Discovery of a temperature-sensitive (ts) mutation affecting the activity of the enzyme provided evidence that the structural protein was HSV-specified (Francke et al., 1978). Preston & Cordingley (1982) mapped the HSV alkaline DNase activity to between and fractional length on the HSV- 1 genome and showed that this region encoded a polypeptide of around mol. wt. (85K). Costa et al. (1983) identified a 2.3 kb mrna mapping between and fractional length on the HSV-1 genome which encoded a polypeptide of 82K that reacted with a monoclonal antibody directed against the HSV-2 alkaline DNase activity described by Banks et al. (1983). The use of deletion mutants confirmed that the lesion affecting the HSV-induced DNase activity maps between and fractional length on the HSV-1 genome (Wathen & Hay, 1984). By a different approach, using monoclonal antibodies directed against the DNase activity, Banks et al. (1985) mapped the active site of this enzyme to between and fractional length on the genomes of both HSV-1 and HSV-2. A ts mutant of HSV-2, ts 13 (Timbury, 1971), was found to possess two distinct mutations, one affecting virion thermostability (Halliburton & Timbury, 1976) and mapping between 0.64 and 0.70 (Chartrand et al., 1981), the other affecting the induction of DNase activity (Francke et al., 1978) and mapping between 0.12 and 0.21 fractional length (Moss et al., 1979). The map position of this latter mutation has since been narrowed to between and fractional length on the HSV-2 genome (data not shown). Revertants of tsl3 able to grow at 38 C had recovered virion thermostability but remained ts for the induction of DNase activity (Moss et al., 1979). This suggested that the HSV DNase activity was non-essential for growth. Francke & Garrett (1982), using one of these revertants (4-8), reported that "viral DNA is synthesized in the absence of active DNase, yet less efficiently so than in its presence" and concluded that the virus-induced enzyme enhanced but was not essential for virus replication. To determine the role, if any, of the HSV DNase activity in virus growth, 4-8 was subjected to more detailed analysis. BHK-21 C13 cells were used throughout this study and were grown in Eagle's medium supplemented with 10 ~ calf serum and 10 ~ tryptose phosphate broth. The wild-type (wt) virus SGM

2 1174 Short communication 6 - (a) I I I 1 I I i I I I (b) (c) I I I I I - 5 X = 4 2 '~ 3 2 g ~o 1 o I i i I I ] o 6 ~Z t, _ (f) i.... ~2 Fig. 1. Time course of induction of HSV-2 DNase activity and of HSV-2 DNA synthesis. Fifty mm plates of BHK-21 cells were mock-infected or infected at 5 p.f.u./cell with HG52 (a, d), 4-8 (b, e), or tsl3 (c, f) virus at 31 C (O), 38.5 C ((3) or 39.2 C (A). At 2 h intervals after infection extracts were prepared and assayed for alkaline DNase activity (a, b, c). No measurable activity was detected in mock-infected cell extracts at any temperature. Virus DNA synthesis (d, e, J3 was measured in successive 2 h periods by labelling with 30 gci [3H]thymidine (sp. act. 25 Ci/mmol) starting at hours 1 to 3 and finishing at hours 11 to 13 post-infection. Virus DNA was separated from host DNA by CsC1 density gradient centrifugation. used was the parental HSV-2 strain HG52; the HSV-1 wt was strain 17. Cell extracts were prepared and assayed for the induction of DNase activity exactly as described previously (Francke et at., 1978). The marker rescue experiments were performed as described previously (Moss et al., 1979) except that transfection was carried out at 37 C rather than 38 C, During the examination of tsl3 and 4-8, it became clear that slight variations in the incubation temperature had a marked effect on the level of virus-induced DNase activity in infected cells. To study the extent of this effect, the level of DNase activity was measured at various times post-infection in cells infected with HG52, tsl3 or 4-8 at three different temperatures (31 C, 38.5 C and 39.2 C) (Fig. 1 a to c). In HG52-infected cells (Fig. 1 a), the level of DNase activity increased linearly at all three temperatures throughout the first 10 h of infection. However, the rate of increase of DNase activity differed depending upon the temperature of incubation, being highest at 38.5 C and lowest at 39-2 C. At 31 C, there was little or no difference in the pattern of induction of DNase activity in cells infected with HG52, 4-8 (Fig. 1 b) or tsl3 (Fig. 1 c) but at 38.5 C, in contrast to wt infection, the level of DNase activity in cells infected with 4-8 and tsl 3 increased only until about 4 h post-infection and then declined; furthermore, the activity found was significantly below that of wt-infected cells at all times. At 39.2 C, only very low levels of virus DNase activity could be detected in 4-8- or tsl 3- infected cells at 2 h whereas virtually none was found at any later time. Preliminary experiments using cells infected with tsl3 or 4-8 had indicated that both synthesized lower amounts of virus DNA at 38 C than at 31 C whereas wt virus synthesized comparable amounts at each temperature (Moss, 1982; Francke & Garrett, 1982). In view of the

3 Short communication 1175 Table 1. Plaque formation and relative plaquing efficiencies (r.p.e.) of HG52 and 4-8 r Titre (p.f.u./ml) R.p.e. A r,k Virus 31 C 38.5 C 39.2 C 31 C/38.5 C 31 C/39.2 C HG x x x x x 108 <1.00 x >3360 effect of temperature on the induction of virus DNase activity by tsl3 and 4-8, experiments were performed to examine HSV DNA synthesis at 31 C, 38.5 C and 39.2 C. In wt-infected cells, virus DNA synthesis commenced between 3 and 5 h after infection at 31 C and reached a maximum between 5 and 7 h (Fig. 1 d); at 38-5 C, virus DNA synthesis had already started between 1 and 3 h post-infection and increased rapidly to a peak between 5 and 7 h which considerably exceeded the peak amount at 31 C; at 39.2 C, DNA synthesis was at a higher level between 1 and 3 h post-infection than at 38.5 C, but the maximum level, reached between 3 and 5 h, was lower than at the other two temperatures. In 4-8-infected cells at 31 C, virus DNA synthesis had already commenced by 3 h post-infection (Fig. 1 e) and reached a peak level between 5 and 7 h which was similar to that after wt infection; at 38.5 C, 4-8 DNA synthesis was considerably higher than at 31 C by 1 to 3 h post-infection and peaked between 3 and 5 h, thereafter declining rapidly to almost zero by 7 h post-infection. In contrast to the wt situation, the total amount of virus DNA synthesis was reduced compared to that at 31 C. The pattern at 39.2 C resembled that found at 38-5 C but the peak level, which had already been attained between 1 and 3 h, was considerably lower and virus DNA synthesis had virtually ceased by 5 h post-infection. The pattern of virus DNA synthesis in tsl3-infected cells essentially resembled that in 4-8-infected cells at all three temperatures (Fig. l f). Virus DNA synthesis during 2 h labelling periods (Fig. 1 d to f) can be contrasted with the level of virus-induced DNase activity present, measured at 2 h intervals (Fig. la to c). At 39-2 C, and to some extent also at 38.5 C, the DNase activity induced after infection of cells with tsl 3 or 4-8 was only present in any significant amount at early times. Although the levels of DNA synthesis do not directly correlate with those of DNase activity (e.g. at 31 C, virus DNase activity increased throughout the period of infection whereas virus DNA synthesis reached a peak level at an early time and then declined), there is clearly a relationship between the patterns obtained for DNase activity and DNA synthesis at the three temperatures. In view of the very low levels of DNase activity and also of virus DNA synthesis in cells infected with 4-8 at 39.2 C, the ability of 4-8 to grow at this temperature was compared with that of HG52. The efficiency of plaquing (e.o.p.) of the wt was similar at 39.2 C and 31 C but the 38.5 C titres were slightly higher (Table 1). With 4-8, the e.o.p, at 38.5 C was tenfold lower than at 31 C, but at 39.2 C there was a dramatic, greater than 1000-fold, reduction in titre (Table 1). Thus, at 39.2 C, in the virtual absence of HSV-induced DNase activity (Fig. 1 b), virus growth was also essentially blocked. Furthermore, the 4-8 plaques which were observed at 39.2 C were very small compared to plaques at 31 C, suggesting that they probably resulted because of leakthrough of the mutant gene product. This temperature-dependent difference in plaque size was not observed with HG52. To determine whether or not the observed effects were consequences of the nuclease lesion of 4-8, marker rescue experiments were performed using the cloned HSV-1 wt EcoRI d fragment which Preston & Cordingley (1982) had shown to direct the synthesis of an HSV-induced alkaline DNase activity following micro-injection into Xenopus laevis oocytes. The marker rescue experiments were carried out by co-transfecting 4-8 DNA with cloned HSV-1 EcoRI d (0.081 to fractional length) into BHK-21 cells at 37 C and the progeny virus was titrated at 31 C, 38.5 C and 39.2 C. The titres from cells transfected with 4-8 DNA were comparable at both 31 C and 38.5 C in the presence or absence of EcoRI d. However, at 39.2 C, the titre of progeny virus from cells receiving 4-8 DNA and EcoRI d was over 470-fold higher than that from those transfected with 4-8 DNA alone (results not shown).

4 1176 Table 2. Comparison of e.o.p., DNA Short communication synthesis, DNase activity and DNA polymerase activity (b) Virus DNA synthesis Incubation (a) Efficiency of plaquing (~ of level at 31 C) temperature c J' ~ r ~ "~ ( C) R13(4-8) HG52 R13(4-8) HG " '55 x l0 s 4.67 x " x 109 1"10 x x t '2 8'90 x 108 <1'00 x 102 5'41 x (c) Virus DNase activity (d) Virus DNA polymerase activity (~ of level at 31 C) (~ of level at 31 C) & F ~ t R13(4-8) HG52 R13(4-8) HG & Plaques from the co-transfection of 4-8 DNA with HSV-1 wt EcoRI d that grew at 39.2 C were isolated and grown into virus stocks at 31 C. Restriction enzyme analysis of the DNA from these stocks showed that they were intertypic recombinants and that they contained HSV-1 DNA inserted in the correct position (data not shown). The results obtained with one such stock [R13 (4-8) 8-3] which was tested together with HG52 and 4-8 virus for e.o.p., virus DNA synthesis and virus DNase activity at 31 C, 38.5 C and 39.2 C, are given in Table 2. The e.o.p. of R 13 (4-8) 8-3 resembled that of wt virus at all three temperatures (Table 2 a). Table 2 (b) shows the level of virus DNA synthesis in cells labelled throughout infection with [3H]thymidine and harvested at 16 h (31 C) or 12 h (38.5 C and 39.2 C) post-infection. These results show that R13 (4-8) 8-3 synthesized wt levels of virus DNA in cells infected at each temperature. Table 2(c) compares DNase activity in cells infected for 8 h; as expected, wt- and R13 (4-8) 8-3-infected cells induced comparably high levels of DNase activity at all three temperatures. H. Moss & B. Francke (unpublished observations) found that, like the alkaline DNase activity, the DNA polymerase activity induced in cells infected with tsl3 was inactivated following a temperature shift from 31 C to 38 C. Subsequently, Francke & Garrett (1982) showed that both tsl 3 and 4-8 induced levels of DNA polymerase activity at 38.5 C which were greatly reduced compared to those of wt. To determine if rescue of the DNase activity resulted in wt levels of DNA polymerase activity, induction of DNA polymerase activity in R13 (4-8) 8-3- infected cells was measured at 8 h post-infection (Table 2d). The results showed a close correlation between the induction of DNA polymerase and DNase activities which indicates that the temperature sensitivity of the tsl 3- and 4-8-induced DNA polymerase activities is a consequence of the DNase defect in these viruses. Previous studies have shown that partially purified DNase from cells infected with tsl 3 or 4-8 is thermolabile in vitro at 45 C (Moss et al., 1979). Analogous experiments with the intertypic recombinant, R13 (4-8) 8-3, demonstrate that rescue of the DNase lesion resulted in the induction of DNase activity which resembled wt virus in thermostability (Fig. 2). To confirm that the above results were a consequence of rescue of the ts DNase lesion by HSV-1 EcoRI d, the polypeptide profiles of cells infected with R13 (4-8) 8-3, HSV-1 wt or HSV-2 wt and labelled with [35S]methionine were compared by polyacrylamide gel electrophoresis (Fig. 3). It is clear from these profiles that R13 (4-8) 8-3 induced a band of 85K which co-migrated with that of HSV- 1, both of which migrated distinctly more slowly than the corresponding HSV- 2 band of 84K (Marsden et al., 1978). Since the only known mutation in 4-8 affects DNase activity, an association between virus DNA synthesis and the induction of DNase activity must be inferred. Our previous study with 4-8 established that the mutation affecting virion thermostability was responsible for the inability of tsl3 to grow at 38 C (Moss et al., 1979). Although virion thermostability had been

5 Short communication 1177! I I I I ---0,..J :,, 75 t~ Z (3 I I i I Prior incubation (min at 45 C) Fig. 2 Fig. 3 Fig. 2. Heat stability of DNase activity in vitro. Partially purified extracts of HG52-, 4-8- and R13 (4-8) 8-3-infected cells were incubated at 45 C as described previously (Francke et al., 1978) at ph 7.5 in the presence of native calf thymus DNA. At various times, from 0 to 120 min, samples were removed and chilled on ice. After removal of the 120 min sample, all samples were assayed for DNase activity. The 100~ value for each extract is the activity at 0 min of incubation at 45 C. II, HG52, 0, 4-8; O, R13 (4-8) 8-3. Fig. 3. -Autoradiograph of potypeptides synthesized in cells infected with R I3 (4-8) 8-3 (lane 1), tsl 3 (lane 2), 4-8 (lane 3), HSV-2 wt (lane 4), HSV-1 wt (lane 5) or mock-infected cells (lane 6) which were separated by electrophoresis through a 7.5~ polyacrylamide gel under denaturing conditions. Cells were infected at 38.5 C and labelled from 5 to 7 h post-infection with [35S]methionine. Cells infected at 31 C and at 39.2 C gave similar results (not shown). The 85K HSV-1 band is indicated by a closed circle and the corresponding HSV-2 band by an open circle, both placed to the left of lane 1. regained by 4-8, the ts DNase lesion of its parent mutant, tsl 3, was retained. This led Moss et al. (1979) and Francke & Garrett (1982) to conclude that the DNase activity was non-essential for virus growth. It was suggested previously (Moss et al., 1979) that this enzyme activity might resemble the HSV-specified thymidine kinase activity which is non-essential for virus growth in rapidly growing BHK-21 cells but is essential for growth in serum-starved 'resting' cells (Jamieson et al., 1974). However, Moss et al. (1979) and Francke & Garrett (1982) conceded that the small amounts of residual DNase activity induced by tsl 3 and 4-8 at 38 C might have been sufficient for normal virus growth. The results presented here demonstrate that it was this residual enzyme activity which allowed normal replication of 4-8 at 38 C. The experiments with 4-8-infected cells make it clear that only a fraction of the DNase activity present in wt-infected cells is necessary to allow normal virus growth and that leak-through of DNase activity at 38 C obscured, in our previous studies, the essential nature of this enzyme. When the temperature of incubation of tsl3- and 4-8-infected cells was increased to 39.2 C, virtually no virus DNase activity was induced apart from low levels at very early times after infection and this was accompanied by substantial reduction in virus DNA synthesis and dramatic loss of virus growth in cells infected with these mutants. The small amounts of virus DNA synthesis observed took place only at early times after infection when low levels of induced DNase activity were detected. Thus, at 38 C and 38.5 C, both tsl3 and 4-8 are only partially temperature-sensitive for the induction of DNase activity and synthesis of virus DNA, but at 39-2 C, enzyme induction and DNA synthesis are both blocked to a far greater extent. Francke & Garrett (1982) observed a 16-fold reduction in titre of 4-8 in cells infected at 38-5 C compared to 31 C: this

6 1178 Short communication result is in agreement with the tenfold reduction in titre found in the present study (Table 1). However, Francke & Garrett (1982) did not check 4-8 at temperatures higher than 38.5 C and thus failed to observe the considerable reduction in titre (> 3360) at 39.2 C (Tabie I). The present study shows that marker rescue of the 4-8 ts lesion with HSV-1 EcoRI d results in wt levels of DNase activity, virus DNA synthesis and virus growth in cells infected at 39.2 C. Furthermore, infection of cells with stocks of the rescued virus results in the induction of DNase activity which exhibits thermostability in vitro resembling that of the wt virus and has the expected HSV-1 DNase migration behaviour. This demonstrates for the first time that the HSV DNase activity provides an essential function for HSV replication. I should like to thank Dr F. J. Rixon for many helpful discussions and Dr C. M. Preston for his encouragement. I am grateful to Professor John H. Subak-Sharpe for his interest in this work and critical reading of the manuscript. 4-8 DNA was a gift from Dr P. Chartrand. The cloned HSV-1 wt EcoRI d fragment was a gift from Dr C. M. Preston. REFERENCES BANKS, L., PURIFOY, D. J. M., HURST, P-F., KILLINGTON, R. A. & POWELL, K. L. (1983). Herpes simplex virus nonstructural proteins. IV. Purification of the virus-induced deoxyribonuclease and characterization of the enzyme using monoclonal antibodies. Journal of General Virology 64, BANKS, L. M., HALLIBURTON, I. W., PURIFOY, D. J. M., KILLINGTON, R. A. & POWELL, K. L. (1985). Studies on the herpes simplex virus alkaline nuclease : detection of type-common and type-specific epitopes on the enzyme. Journal of General Virology 66, CHARTRAND, P., WILKIE, N. M. & TIMBURY, M. C. (1981). Physical mapping of temperature-sensitive mutations of herpes simplex virus type 2 by marker rescue. Journal of General Virology 52, COSTA, R. H., DRAPER, K. G., BANKS, L. POWELL, K. L., COHEN, G., EISENBERG, R. & WAGNER, E. K. (1983). Highresolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein. Journal of Virology 48, FRANCKE, B. & GARRETT, B. (1982). The effect of a temperature-sensitive lesion in the alkaline DNase of herpes simplex virus type 2 on the synthesis of viral DNA. Virology 116, FRANCKE, B., MOSS, H., TIMBURY, M. C. & HAY, J. (1978). Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2. Journal of Virology 26, HALLIBURTON, I. W. & TIMBURY, M. C. (1976). Temperature-sensitive mutants of herpes simplex virus type 2: description of three new complementation groups and studies on the inhibition of host cell DNA synthesis. Journal of General Virology 30, HAY, J., MOSS, H. & HALLIBURTON, I. W. (1971). Induction of deoxyribonucleic acid polymerase and deoxyribonuclease activities in cells infected with herpes simplex virus type II. Biochemical Journal 124, 64. JAMIESON, A. T., GENTRY, G. A. & SUBAK-SHARPE, J. H. (1974). Induction of both thymidine and deoxycytidine kinase activity by herpes viruses. Journal of General Virology 24, KEIR, H. M. & GOLD, E. (1963). Deoxyribonucleic acid nucieotidyltransferase and deoxyribonuclease from cultured cells infected with herpes simplex virus. Biochimica et biophysica acta 72, MARSDEN, H. S., STOW, N. D., PRESTON, V. G., TIMBURY, M. C. & WILKIE, N. M. (1978). Physical mapping of herpes simplex virus-induced polypeptides. Journal of Virology 28, MOSS, H. W. McL. (1982). The herpes simplex virus specified deoxyribonuclease and DNA polymerase activities. Ph.D. thesis, University of Glasgow. MOSS, H., CHARTRAND, P., TIMBURY, M. C. & HAY, J. (1979). Mutant of herpes simplex virus type 2 with temperaturesensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc- lesion Journal of Virology 32, PRESTON, C. M. & CORDINGLEY, M. G. (1982). mrna- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes. Journal of Virology 43, TIMBURY, M. C. (1971). Temperature-sensitive mutants of herpes simplex virus type 2. Journal of General Virology 13, WATHEN, M. W. & HAY, J. (l 984). Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones. Journal of Virology 51, (Received 5 December 1985)