Journal o f General Virology (1992), 73, 1417 1428. Printed in Great Britain

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Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation C. C. Smith, 1,3. M. Kulka, ~ J. P. Wymer, 1 T. D. Chung 1 and L. Aurelian 1,2,3 1Virology/Immunology Laboratories, Department of Pharmacology and Experimental Therapeutics, The University of Maryland School of Medicine, Baltimore, Maryland 21201 and Departments of 2Comparative Medicine and 3Biochemistry, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, U.S.A.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP 10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of 1CPI0 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 °C compared to 34 °C after 12 h post-infection. The efficiency of plaquing is 0-003. The growth defect at 39 °C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations

resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP 10 gene from tsS-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 °C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 °C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors p J W l 7 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.

Introduction

amino-terminal domain with properties similar to those of growth factor receptor kinases (Chung et al., 1989, 1990) and which have transforming potential (Yarden & Ullrich, 1988). DNA sequences that encode the ICP10 protein kinase (PK) domain transform immortalized ceils to a neoplastic phenotype (Jariwalla et al., 1980; Hayashi et al., 1985; Iwasaka et al., 1985). Recent studies using an ICP6 deletion mutant or a lacZ insertion mutant have shown that R R activity is required for HSV-1 growth in non-dividing cells or in cells infected at 39 °C (Goldstein & Weller, 1988a, b; Preston et al., 1988). However, the role that ICP10 gene expression may play in the HSV-2 life cycle and/or its neoplastic potential is not yet clearly understood. To address these questions we used a spontaneous mutant that is temperature-sensitive in ICP 10 synthesis (ts 5-152) and ICPI0 expression vectors whose construction has

Ribonucleotide reductase (RR) is involved in a major pathway for the production of DNA precursors, thereby playing a key role in D N A synthesis of eukaryotic and prokaryotic cells. Like the mammalian and bacterial enzymes, the herpes simplex virus (HSV) RR consists of two non-identical subunits. The large subunit (RR1, Mr 140K) designated ICP6 and ICP10 for HSV-1 and HSV-2 respectively, is encoded within the UL region of the viral genome (UL39 for HSV-1). The small subunit (RR2, Mr 38K) is encoded by a 1-2 kb m R N A (UL40 for HSV-1) that overlaps the 3' end of the 5.0 kb transcript of the large subunit (Anderson et al., 1981 ; McLauchlan & Clements, 1983; Bacchetti et al., 1984; Frame et al., 1985). However, unlike the mammalian and bacterial enzymes, the HSV RR1 contains an additional 57K 0001-0779 © 1992 SGM

1418

C. C. Smith and others

been described previously (Chung et al., 1989). Our findings indicate that ICP10 expression is essential for HSV-2 growth and neoplastic potential. Methods Cells and viruses. HEp-2 cells were grown in medium 199 with 10~ calf serum. Primary rabbit kidney cells (Whitaker Bioproducts) and human lung fibroblast MRC-5 were grown in Eagle's MEM with 10~ foetal calf serum (FCS). Normal diploid Syrian hamster embryo (SHE) cells and SHE cells immortalized with the AE fragment of HSV-2 DNA (AE cells; Jariwalla et al., 1983) were grown in IBR-modified Dulbecco Eagle's reinforced medium (ERM) supplemented with 10~ FCS (Manak et al., 1981 ; Hayashi et al., 1985). The (G) and (ANG) strains of HSV-2 were grown as described (Manak et al., 1981). The tsH9 mutant, defective in the major DNA-binding protein (Spang et al., 1983), was obtained from Dr P. Schaeffer (Harvard University, Boston, Mass., U.S.A.). The 859 virus was isolated from a genital lesion and was serotyped as HSV-2 (Aurelian et al., 1990). Virus titres (p.f.u.) were determined at 34 °C and 39 °C under a methyl cellulose overlay (Aurelian, 1968). Relative growth expressed as efficiency of plaquing (e.o.p.) represents p.f.u, at 39 °C/p.f.u. at 34 °C. Plasmids. Plasmid pGH17a contains the entire HSV-2 gene for the ICP10 protein (coding and promoter; Iwasaka et al., 1985). The

construction of the ICP10 constitutive expression vector pJWl7, the amino-terminal construct that expresses the ICP 10 PK domain pJW32, and the frameshift mutant pJW21 was previously described (Chung et al., 1989). The plasmid pJW31 was constructed from pJW17 by B a l i and Stul digestion and religation to remove the 1.3 kb fragment that encodes the ICP10 PK domain (Chung et al., 1989). Antibodies. The preparation and specificity of monoclonal antibodies (MAbs) 30, G8 and 27 that recognize ICP10 (MAbs 30 and G8) and the HSV-2 major DNA-binding protein ICSP11/12 (MAb 27), respectively have been described (Aurelian et al., 1989; Chung et al., 1989, 1991). MAb 18etA5 obtained from Dr N. Balachandran (Kansas City, Kan., U.S.A.) is specific for the HSV-2 glycoprotein gG-2 (Roizman et al., 1984). Immunofluorescence. Slides were fixed in cold methanol ( - 20 °C) at post-treatment population doublings (PTPD) 2, 13 and 26. They were stained with MAb 30 (1 h, 37 °C) followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1 h, 37 °C) as described (Wymer et al., 1989). Preparation o f radiolabelled cell extracts and immunoprecipitation.

Cells were labelled with [35S]methionine (50 ~tCi/ml, sp. act. 1120 Ci/mmol; NEN Research Products) in MEM containing one-tenth the normal concentration of methionine and 1 ~ dialysed FCS. Extracts were prepared in cold radioimmunoprecipitation buffer (10mM-TrisHcl pH 8.0, 1 ~ deoxycholate, 0.1~ SDS, 1 ~ NP40, 0-15 M-NaC1, 1 mM-DTT, 1 mM-PMSF), disrupted by sonication and clarified by centrifugation (16000 g for 30 rain). Proteins were precipitated by incubation (1 h at 4 °C) with antibody and (30 rain at 4 °C) with Protein A-Sepharose CL4B (Sigma), resolved by 8.5~ SDS-PAGE and quantified by densitometric scanning (Chung et al., 1989). Cell fractionation was as described (Chung et al., 1989). R R assay. Cell extracts (in 20 mM-HEPES buffer pH 7.2, with 2 mMDTT) were clarified by centrifugation (100000 g, for 60 min at 4 °C) and treated with ammonium sulphate (45~ saturation). Following dialysis and centrifugation (16000 g for 30 min) they were incubated (10 min at 37 °C) with equal volumes of a double-strength standard reaction mixture containing 400 mM-HEPES pH 8-0, 20 mr,l-DTT and

0.2 mM-[3H]CDP (20 mCi/mmol, NEN). The reaction was terminated by the addition of 100 mM-hydroxyurea with 10 mM-EDTA (pH 8-0) and boiling for 3 min. Crotalus atrox venom (Sigma) was added (0.5 mg/ml in 12 mM-Tris pH 9-0, 4 mM-MgC12, 1 mM-deoxycytidine) and the mixture was incubated for 30 min at 37 °C, boiled for 3 min and applied to a 0.5 ml Dowex-1 borate column. The column was washed with 2.5 ml H,O and 0.5 ml eluate fractions were mixed with 5 ~tl of Opti-Fluor (Packard Instrument Company) for scintillation counting. Reductase activity is expressed as units/mg protein, where 1 unit represents the conversion of 1 nmol [3H]CDP to dCDP per hour (Huszar & Bacchetti, 1981). D N A polymerase assay. Viral DNA polymerase activity was assayed as described (Powell & Purifoy, 1977), on high-salt extracts of HSV-2infected (20 p.f.u./cell; 18 h p.i.) or mock-infected (PBS at pH 7.2) HEp-2 cells. The standard assay mixture containing 8.8 mM-Tris-HCl pH 7.5, 2.6 mM-2-mercaptoethanol, 4 mM-MgCI2, 133 mM-NH4SO4, 0-25 mM-dCTP, -dGTP and -dATP, 0-4 pM-[3H]TTP (81.6 Ci/mmol, NEN Research Products), 100 lag partially degraded salmon sperm DNA and 50 lal of enzyme extract in a 200 ~tl volume was incubated for 30 min at 39 °C. The reaction was terminated by addition of 20 ~ TCA acid with 0.25 mg/ml of BSA, and the acid-insoluble material was collected on glass fibre filters, air-dried and counted for radioactivity. Immune complex kinase assay. Unlabelled cell extracts were standardized for protein concentration and immunoprecipitated with antibody and Protein A-Sepharose CL4B. The beads were washed three times with radioimmunoprecipitation buffer and three times with TS buffer (20 mM-Tris-HC1 pH 7-4, 0.15 ~-NaCI), suspended in 50 ptl of kinase reaction buffer consisting of 10 laCi of [7-32p]ATP (0.1 laM; specific activity 3000 Ci/mmol; NEN Research Products), 5 mMMgCI2, and 20 mM-Tris-HC1 pH 7.4, with or without 5 pg of histone type III-S (from calf thymus; Sigma) and incubated at 30 °C for 10 min. The reaction was terminated by boiling in 100 lal of x 0.5 denaturing solution, and the proteins were resolved by SDS-PAGE (Chung et al., 1989). D N A transfection. Cells (1 × 105/well) were plated 24 h prior to transfection into six-well cluster dishes (Costar) and transfected with supercoiled plasmid DNA (2-5 lag/well) by the calcium phosphate precipitation method using a glycerol boost (Wymer et al., 1989; Chung et al., 1989). They were harvested or passaged 40 to 44 h posttransfection. D N A sequencing and analysis. DNA extracted from ts5-152 cytoplasmic virions (Pignatti et al., 1979) was digested with B a m H I and electrophoresed on 0.8~ agarose gels in 90 mM-Tris-borate/l mraEDTA buffer. Fragments in the 6-0 to 9-0 kb range were electroeluted, purified by two cycles of phenol-chloroform extraction and ligated into pUC19. A clone (pMK 1) was isolated that contained a 7.6 kb fragment hybridizing with the SalI C [map unit (m.u.) 0-559 to 0-567] and S a i l D (m.u. 0.555 to 0.559) fragments from pGH17a. The PstI B and PstI C fragments of p MK 1 were digested with S a i l and cloned into M 13mp 18 and M13mpl9. The DNA sequence was identified by the Sequenase (US Biochemicals) dideoxynucleotide chain termination method (Sanger et al., 1977). Sequence data were compiled and analysed with the PC Gene program (Intelligenetics). Cloning o f the ts5-152 ICPIO gene into wild-type (wt) virus 859. Viral DNA was extracted from 859 cytoplasmic virions by NaI density gradient centrifugation (Pignatti et al., 1979). It was checked for purity by digestion with B a m H I and electrophoresis on a 0.8~ agarose gel. A 4.4 kb DNA fragment, containing the ICP10 gene, was purified from the pMK1 clone by double digestion with B a m H I and SacI. After electrophoresis on a 0.8 ~ agarose gel, the 4.4 kb fragment was excised, electroeluted and purified by two cycles of phenol-chloroform extraction prior to ethanol precipitation at - 20 °C. Rabbit kidney cells were

H S V - 2 ribonucleotide reductase large subunit

cotransfected with infectious 859 DNA and the 4.4 kb DNA fragment from pMK 1. The transfection mixture contained 1 f.tgof 859 DNA, 2 ktg of the 4.4 kb DNA fragment and 15 ~tg/ml salmon sperm DNA. Transfections were done as described (Knipe et al., 1979) except that cells were overlaid with MEM with 2% FCS and incubation was at 34 °C. When generalized c.p.e, was observed, virus was harvested and plaque-purified under a methyl cellulose overlay (Aurelian, 1968) at 34 °C. Virus from individual plaques was grown for one passage in HEp-2 ceils prior to titration at 34 oC and 39 °C.

Establishment of transformed lines and anchorage-independent growth. Transformation with u.v.-inactivated virus was done as described (Manak et al., 1981). SHE cells were exposed to the equivalent of 1 p.f.u./cell of inactivated virus and serially passaged at split ratios of 1 : 5 to 1 : 10. Control cultures died out within 18 to 42 PTPD which was calculated based on generation times of 14 and 12 h for normal and transformed SHE cells respectively (Barrett et al., 1979). Cultures surviving exposure to inactivated virus developed into established lines (Manak et al., 198 l). Transformation by ICP 10 expression vectors was studied in AE cells because ICP10-encoding DNA sequences transform immortalized but not diploid cells (Jariwalla et al., 1983; Hayashi et al., 1985). Cells were serially passaged (1:10 to 1:20 split ratios) beginning at 40 to 44 h post-transfection and assayed for anchorageindependent growth at PTPD 13 to 26. For anchorage-independent growth, cells (500/ml) were resuspended in ERM-10% FCS with 0-5% Sea-Plaque agarose (FMC) and 0-1% peptone and layered on top of a solidified basal layer (5 ml) of ERM-10% FCS containing 1% agarose and 0.2% peptone. After 14 to 21 days at 37 °C, large (>0.2 mm) colonies that correlate with tumorigenic potential in neonatal hamsters (Jariwalla et al., 1983, Hayashi et al., 1985) were scored using a calibrated microscope eyepiece. Cloning efficiency is expressed as the number of colonies ( x 100)/number of seeded cells (Manak et al., 1981 ; Jariwalla et al., 1980, 1983; Hayashi et at., 1985).

Results Isolation and temperature sensitivity o f ts5-152 virus Virus ( L F V ) isolated f r o m a genital lesion a n d s e r o t y p e d as H S V - 2 ( A u r e l i a n et al., 1990) s h o w e d a lower level o f r e p l i c a t i o n on M R C - 5 a n d H E p - 2 cells at 39 °C as c o m p a r e d to 3 4 ° C (e.o.p. 0.09; T a b l e 1) a n d was s u b m i t t e d to p l a q u e p u r i f i c a t i o n a f t e r one initial p a s s a g e in H E p - 2 cells. T e n p l a q u e - p u r i f i e d isolates were a s s a y e d for g r o w t h on H E p - 2 cells at 34 °C a n d 39 °C. A l l f o r m e d p l a q u e s e q u a l l y well at 34 °C. H o w e v e r , t h e y s h o w e d a r e l a t i v e l y wide s p e c t r u m o f p l a q u i n g efficiencies at 39 °C. E.o.p. values r a n g e d f r o m 0.23 for isolate 5-1 to 0-004 for isolate 5-15 ( T a b l e 1). T h e 5-15 isolate was serially p a s s a g e d in H E p - 2 cells a n d p l a q u i n g efficiency at b o t h t e m p e r a t u r e s was d e t e r m i n e d at p a s s a g e 10, a n d a g a i n after two a d d i t i o n a l cycles o f p l a q u e purification. T h e isolate o b t a i n e d after t h e s e last t w o cycles o f p l a q u e purification, d e s i g n a t e d ts5-152, h a d a n e.o.p, value o f 0-003 w h i c h was s i m i l a r to t h a t o f the 5-15 p l a q u e isolate. T h i s c o m p a r e s to e.o.p, values o f 1-10, 1.07 a n d 1-0 for wt strains G , A N G a n d 859 r e s p e c t i v e l y ( T a b l e 1). W e i n t e r p r e t these findings to i n d i c a t e t h a t t h e o r i g i n a l

1419

Table 1. Plaquing efficiencies o f H S V - 2 plaque-purified isolates at 34 °C and 39 °C P.f.u./ml Virus

34 °C

39 °C

E.o.p.*

LFV t 5-15 5-15~ ts5-152§ HSV-2 (G) HS¥-2 (ANG) 859 859/152

7-4 × 106 3 × 10v 5 x 108 3-2 x l0 s 7-3 × 108 7-I x l0T 2-2 × 106 8-8 × 106

7.2 x 105 6.9 × 106 1.8 x 106 1.1 × 106 8.1 x 108 7-6 × 107 2.2 x 106 1.7 x 10a

0.09 0.23 0-004 0.003 1.10 1.07 1.0 0-002

* Plaquing efficiencies were determined by titrating virus stocks on monolayers of HEp-2 cells at the indicated temperature. E.o.p. is expressed as p.f.u, at 39 °C/p.f.u. at 34 °C. t Original HSV-2 isolate from a genital lesion (Aurelian et al., 1990). Plaque-purified isolates of LFV. § The 5-15 isolate was seriallypassaged (10 times) on HEp-2 cells and subjected to two additional cycles of plaque purification. The resulting isolate is designated ts5-152.

L F V isolate is h e t e r o g e n e o u s , c o n s i s t i n g o f d i s t i n c t virus p o p u l a t i o n s t h a t are c o m p r o m i s e d differently for p l a q u i n g efficiency at 39 °C. M u t a n t ts5-152, w h i c h is significantly c o m p r o m i s e d , is stable a n d t h e r e f o r e used t h r o u g h o u t these studies. T h e effect o f t e m p e r a t u r e on the a b i l i t y o f ts5-152 to p r o d u c e infectious virus was d e t e r m i n e d by single-step g r o w t h analysis at 34 °C a n d 39 °C. Y i e l d s o f p r o g e n y virus at e a c h t i m e p o i n t were d e t e r m i n e d on H E p - 2 cells at 34 °C. Virtually i d e n t i c a l g r o w t h p a t t e r n s were seen for ts5-152 a n d H S V - 2 ( G ) at 34 °C; H S V - 2 ( G ) g r e w equally well at 39 °C. H o w e v e r , t h e titres o f ts5-152 p a r a l l e l e d those o f H S V - 2 (G) o n l y until 12 h p.i. T h e y d i d n o t i n c r e a s e b e y o n d this time, a n d by 48 h p.i. t h e y were 1000-fold l o w e r t h a n those seen at 34 °C (Fig. 1).

Effect o f temperature on ts5-152 protein synthesis T o d e t e r m i n e t h e effect of t e m p e r a t u r e on ts5-152 p r o t e i n synthesis, H E p - 2 cells were i n f e c t e d w i t h ts5-152 at 34 °C or 39 °C a n d labelled w i t h [35S]methionine f r o m 2 to 12 h p.i., a n d p r o t e i n s were s e p a r a t e d b y S D S P A G E . H S V - 2 ( G ) - i n f e c t e d cells were used as control. V i r t u a l l y i d e n t i c a l p r o t e i n profiles were seen for H S V - 2 (G) at 34 °C a n d 39 °C (Fig. 2b) a n d for ts5-152 at 34 °C (Fig. 2 a , l a n e 1). H o w e v e r , in cells i n f e c t e d w i t h ts5-152 at 39 °C the levels o f a 140K p r o t e i n c o n s i s t e n t w i t h I C P 1 0 were /> 10-fold lower (Fig. 2a, l a n e 2). A l l o t h e r p r o t e i n s were s y n t h e s i z e d e q u a l l y well at b o t h t e m p e r a tures a n d t e m p e r a t u r e h a d n o effect on t h e p r o t e i n profile o f m o c k - i n f e c t e d cells (Fig. 2c).

C. C. Smith and others

1420

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I I 24 48 Time p.i. (h) Fig. 1. Single-step growth curves. Monolayers of HEp-2 cells were infected with 5 p.f.u./cell of HSV-2(G) at 34 °C (O) or 39 °C (O) or with ts5°152 at 34 °C (A) or 39 °C (A) and virus titres were assayed at 2 to 48 h p.i. (a)

(b) 1

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Fig. 2. Effect of temperature on the synthesis of infected cell proteins. HEp-2 cells were infected at 34 °C (lanes 1) or 39 °C (lanes 2) with ts5152 (a), HSV-2(G) (b), or mock-infectedwith PBS (c) and labelled with [35S]methionine from 2 to 12 h p.i. Proteins were resolved by SDS-PAGE.

Immunoprecipitation with M A b s specific for ICP10, or for two unrelated proteins (gG-2 and I C S P l l / 1 2 ) , confirmed the interpretation that only I C P I 0 synthesis was significantly decreased in cells infected with ts5-152 at 39 °C. The levels of ICP10 precipitated by M A b 30 (Fig. 3 b, lanes 5 and 6) or M A b G8 (Fig. 3 b, lanes 7 and 8) from cells infected with ts5-152 at 39 °C (and labelled with [3SS]methionine at 2 to 12 h p.i.) were >/10-fold lower than those precipitated from ts5-152-infected cells at 34 °C. Equal levels of ICP10 were precipitated from ceils infected with the wt strains G (Fig. 3c) or A N G (Fig. 3a, lanes 2 and 3), or with an unrelated mutant (tsH9) (Fig. 3b, lanes 3 and 4) at 34°C and 39°C. Consistent with the relatively higher e.o.p, of the 5-1 as compared to the ts5-152 isolate (Table l), the levels of ICP10 precipitated from cells infected with 5-1 at 39 °C (Fig. 3b, lane 1) were only two- to threefold lower than those from 5-l-infected cells at 34 °C (Fig. 3b, lane 2). Virtually identical levels of gG-2 (Fig. 4, lanes 4 and 5) and I C S P I 1/12 (Fig. 4, lanes 1 and 2) were respectively precipitated by M A b s 18etA5 and 27, from cells infected with ts5-152 at 34 °C and 39 °C. Temperature had no effect on the intracellular localization of I C P I 0 in ts5-152-infected cells. The highest levels were in the soluble fraction (Chung et al., 1989) and were similar at 34 °C and 39 °C (82.7 + 2 ~ and 64 + 11 ~ respectively). Cytoskeletal localization was 11.9 +_ 8 ~o at 34 °C, and 33 +_ I ~o at 39 °C. The somewhat higher level at 39 °C probably reflects technical variability as a similar increase was seen also for an unrelated 60K protein (28 + 7 ~ and 44 _+ 3 ~ at 34 °C and 39 °C respectively).

RR, ICPIO P K and DNA polymerase activities in ts5-15 2-infected cells H E p - 2 or AE cells mock-infected with PBS or infected with 20 p.f.u./cell of ts5-152 HSV-2 (G) or ( A N G ) were assayed for R R and ICP10 P K activities at 12 h p.i. D N A polymerase activity was assayed at 18 h p.i. since previous findings had indicated that in HSV-2-infected cells D N A polymerase activity is maximal at 12 to 24 h p.i. (Powell & Purifoy, 1977). R R activity was similar in cells infected with HSV-2 (G) or ( A N G ) at 34 °C or 39 °C. However, consistent with the significantly lower levels o f l C P 1 0 in cells infected with ts5-152 at 39 °C, R R activity was virtually undetectable at this temperature (Table 2). The levels of ICP10 P K activity determined by autophosphorylation or histone transphosphorylation (Chung et al., 1989) were also significantly (>/10-fold) lower in AE or H E p - 2 cells infected with ts5-152 at 39 °C (Fig. 5 a, lanes 6 and 8) as compared to 34 °C (Fig. 5 a, lanes 4 and 7). Both auto- and transphosphorylating activities were similar in AE or H E p - 2 cells infected with

H S V - 2 ribonucleotide reductase large subunit

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1421

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: :..:::: 2::): 34 39 34 39 34 39 39 34 34 39 34 39 Fig. 3. Effect of temperature on ICP10 synthesis. (a) MAb 30 immunoprecipitate of extracts from HEp-2 cells infected with HSV-2 (ANG) and labelled with [35S]methionine at 2 to 12 p.i. at 34 °C (lane 2) or 39 °C (lane 3). Total extract proteins are shown in lane 1, (b) MAb 30 immunoprecipitate of extracts from HEp-2 cells infected with plaque isolate 5-1 at 39 °C (lane 1) or 34 °C (lane 2), with tsH9 at 34 °C (lane 3) or 39 °C (lane 4) or with ts5-152 at 34 °C (lane 5) or 39 °C (lane 6). MAb G8 immunoprecipitate of extracts from HEp-2 cells infected with ts5-152 at 34 °C (lane 7) or 39 °C (lane 8), (c) MAb 30 immunoprecipitate of extracts from HEp-2 cells infected with HSV-2 (G) at 34 °C (lane 1) or 39 °C (lane 2).

Table 2. Viral R R and D N A polymerase activities in ts5-152-infected cells at 34 °C or 39 °C RR specific activity* (units/rag protein)

DNA polymerasespecific activity~ (units/rag protein)

Extract

34 °C

39 °C

34 °C

39 °C

HSV-2 (G) HSV-2 (ANG) ts5-152 Mock

20.1 19.3 16"2 2-4

18.1 16.9 1"8 2.1

58.6 ND:~ 63"8 16.4

55 ND 51 15.8

* One unit of RR represents the conversion of 1 nmol CDP to dCDP/h. i One unit of DNA polymerase activity represents 1 nmol of TTP incorporated/h. ~:ND, Not determined.

H S V - 2 ( A N G ) at 34 °C (Fig. 5a, lane 3 a n d b, lane 1), o r 39 °C (Fig. 5a, lane 5 a n d b, lane 2), in H E p - 2 cells i n f e c t e d with t s H 9 at 34 °C (Fig. 5e, l a n e 2) or 39 °C (Fig. 5c, lane 1) or in H E p - 2 cells i n f e c t e d w i t h H S V - 2 ( G ) at

3 4 ° C (Fig. 5d, lane 1) or 3 9 ° C (Fig. 5d, lane 2). P h o s p h o r y l a t e d species were not d e t e c t e d in c o m p l e x e s f o r m e d with M A b 27 (specific for I C S P l l / 1 2 ) f r o m H S V - 2 ( A N G ) - (Fig. 5b, lanes 3 a n d 4) or H S V - 2 (G)(Fig. 5a, lane 2) i n f e c t e d cells. D N A p o l y m e r a s e a c t i v i t y was s i m i l a r for ts5-152 a n d H S V - 2 (G) at 34 °C a n d 39 °C ( T a b l e 2). Sequence o f the ts5-152 I C P I O gene Several p o i n t m u t a t i o n s in the p r o m o t e r r e g i o n of ts5-152 as c o m p a r e d to the 333 s t r a i n of H S V - 2 ( S w a i n & G a l l o w a y , 1986) w e r e identified, i n c l u d i n g t r a n s v e r s i o n m u t a t i o n s at p o s i t i o n s - 512 (A to T), - 500 (C to G ) a n d - 285 (C to G ) relative to t h e t r a n s c r i p t i o n s t a r t site a n d t r a n s i t i o n a l m u t a t i o n s at p o s i t i o n s - 4 3 5 (A to G ) a n d + 3 (C to T). T h r e e insertion m u t a t i o n s were seen a t p o s i t i o n s - 3 4 9 , - 3 3 2 a n d - 2 3 0 a n d four d e l e t i o n s at p o s i t i o n s - 275, - 274, - 217 a n d + 75 ( d a t a not shown). T h e s e base c h a n g e s a p p e a r c o n s e r v a t i v e a n d are n o t located within known regulatory or enhancer sequences ( W y m e r et al., 1989; Jones, 1989). O n the o t h e r h a n d , a

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C. C. Smith and others 1

2

4

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two transitions at nucleotide positions 891 (T to C) and 1060 (A to G) and one nucleotide transversion at position 1134 (C to G) resulting in a Ser to Gly substitution at amino acid residue 309 (Fig. 6). The Gly mutation may decrease protein folding stability as Gly has conformational flexibility (Matthews et al., 1987).

5

]CSP11/12 ~ l gG-2

Thermostability of the ts5-152 ICPIO protein

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These experiments were designed to determine the effect of the ICP10 mutation on the metabolic stability of the protein. HEp-2 cells infected with ts5-152 were pulselabelled for 10 rain at 8 h p.i. at 34 °C and harvested immediately after the pulse or after an additional 6 h of incubation at 34 °C or 39 °C in medium containing 400fold excess unlabelled methionine and 50 ~g/ml cycloheximide (chase). ICP10 was precipitated by MAb 30. As shown in Fig. 7, the levels of ICP10 remained stable or were only minimally reduced (~