tims Research, 26 (1992) 141-152 0 1992 Elsevier Science Publishers
VIRUS
141 B.V. All rights reserved
016%1702/92/$05.00
00834
The herpes simplex virus type 1 ICP6 gene is regulated by a ‘leaky’ early promoter * Ping Sze and Ronald
C. Herman
Syntev Research, Palo Alto, CA 94304, USA (Received
18 May 1992; revision
received
and accepted
3 August
1992)
Summary
Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the ZucZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICPO as the major transactivator of the ICP6 promoter. Herpes simplex virus; Gene expression; ICP6; ICPO
Introduction
Three classes of herpes simplex virus (HSV) genes have been recognized on the basis of the timing and regulation of their expression after infection (Honess and Roizman, 1974, 1975). The five immediate early (IE) (or alpha) genes are expressed immediately after infection and are transcribed even in the presence of a high concentration of the protein synthesis inhibitor cycloheximide. Expression of Correspondence to: R.C. Herman, Syntex Research, 3401 Hillview Avenue, Fax: (1) (415) 354-7363, after 28 December 1992: (1) (415) 796-7363. * Manuscript no. 351 from the Institute of Bio-Organic Chemistry.
Palo Alto, CA 94304, USA.
142
the IE genes is dependent upon the interaction of the virion tegument protein known as the alpha-transinducing factor (TIF; alternatively called Vmw65, VP16, ICP25, or UL48) with the POU domain cellular transcription factor Ott-1 (Kristie and Roizman, 1987, 1988; O’Hare and Goding, 1988; O’Neill et al., 1988; Sturm et al., 1988; Kristie and Sharp, 1990) and other cellular proteins (M&night et al., 1987; Gerster and Roeder, 1988; O’Hare et al., 1988; Preston et al., 1988; Triezenberg et al., 1988; Greaves and O’Hare, 1989, 1990; Kristie et al., 1989; Stern et al., 1989; Haigh et al., 1990; Xiao and Capone, 1990). Genes responsive to transactivation by TIF have 5’. . . TAATGARAT.. .3’ or related consensus sequence (Mackem and Roizman, 1982; Spector et al., 1990, 19911, or 5’. . . GCGGAA.. .3’ consensus sequence (Triezenberg, 1988) in the upstream regulatory region. Early (beta) genes require the presence of one or more of the IE proteins for normal expression. Late (gamma) genes are expressed after the onset of DNA replication, and can be further subdivided into a group that requires DNA synthesis for expression and a group that does not. ICP6 (UL39) encodes the large subunit (RRl) of the HSV-1 ribonucleotide reductase that is important for viral DNA synthesis as well as for neurovirulence and reactivation in several animal models (Cameron et al., 1988; Jacobson et al., 1989; Turk et al., 1989; Idowu et al., 1992). It has been suggested that ICP6 is an IE gene because its mRNA can be detected after infection in the presence of cycloheximide (Honess and Roizman, 1974; Watson et al., 1979). Others have shown that ICP6 and the HSV-2 equivalent ICPlO are responsive to TIF in transient expression assays perhaps because of the presence of an upstream TAATGARAT-like element. Transient assays also suggest that the promoter is responsive to ICPO in some cell lines but it is apparently not responsive to ICP4 (Goldstein and Weller, 1988b; Wymer et al., 1989). We report here the quantitative results of experiments designed to examine the regulation of the ICP6 gene in infected Vero cells and in a stably transfected cell line. These results indicate that the ICP6 promoter is weakly transactivated by TIF and maximally induced by ICPO, but is not activated by ICP4.
Materials
and Methods
Cells and Liruses
Vero cells were grown in minimal essential medium supplemented with 5% fetal calf serum and non-essential amino acids. The G5 cell line is a stably transfected Vero clone that carries the HSV-1 TIF gene under control of the inducible mouse metallothionein (mt-1) promoter (plasmid pRB3522 obtained from B. Roizman) plus the same ICP6::lacZ fusion that was inserted into the HSV-1 de122Z mutant (see below); the cells were grown in media supplemented as above plus G418 (500 pgg/ml; GIBCO). Where indicated, expression of TIF was induced by incubating the cells for 18 h in media supplemented as above plus 120 PM ZnSO,. De122Z is a deletion mutant of HSV-l(F) in which the coding sequence for ICP22 (Usl) was replaced by the ICP6 promoter linked to the bacterial 1acZ gene.
143
In this const~ction, 1.19 kb of upstream sequence, including the codons for the first 60 amino acids of ICP6, were fused in frame to 1ucZ (Goldstein and Weller, 1988b). A detailed description of the de122Z mutant will be presented elsewhere (Poffenberger et al., in press). HSV-l(KOS) RH105 is a recombinant virus (obtained from E. Mocarski) that carries the E. co/i 1ucZ gene under the control of the ICP4 promoter and inserted into the HSV thymidine kinase gene (Ho and Mocarski, 1988). HSV-l(KOS) hrR3 is a recombinant virus (obtained from S. Weller) that has the la& gene inserted in-frame directly into the ICP6 RR1 coding sequence following the codon for amino acid 434 (via the BarnHI site at map coordinate 0.574) (Goldstein and Weller, 1988a). HSV-l(KOS) n428 is a nonsense mutant (obtained from P. Schaffer) that contains a synthetic nonsense N&I linker inserted into the ICPO coding sequence and disrupting the reading frame at amino acid 428 (Cai and Sehaffer, 1989). HSV-l(KOS) d120 is a deletion mutant (obtained from P. Schaffer) from which the coding sequence for ICP4 was deleted (DeLuca et al., 1985). Infections and cycloheximide reversal conditions
The cells were infected in the absence or presence of ~cloheximide (100 fig/ml) at a multipli~i~ of infection (MOI) of 10. Virus was adsorbed for 1 h at 37°C; the inoculum was removed and replaced with fresh warm media, and the cells were incubated at 37°C. Extracts were prepared from cells infected in the absence of cycloheximide at 6 h postinfection (p.i.>. When cycloheximide was used, the cells were pretreated for 30 min and then infected in the presence of the drug. The media containing ~ycloheximide was removed at 6 h p.i.; the cells were washed three times with media containing actinomycin D (5 pg/ml). The infection was allowed to proceed in the presence of actinomycin D. Extracts of cells infected under the reversal conditions were prepared 4 h after removal of the cycloheximide (10 h p.i.). zeta-ga~actosidase ~~-ga~~assay
Beta-gal activity was determined essentially as previously described (Spaetc and Mocarski, 1985). Cells in lOO-mm dishes were washed with cold phosphate-buffered saline (PBS), scraped into 1 ml of PBS, and suspended in 5 ml PBS. Cells were pelleted, resuspended in 100 ~1 of freeze-thaw buffer (0.25 M sucrose, 10 mM Tris-HCI, pH 7.4, 10 mM EDTA) and alternately frozen (-SO*C> and thawed (37°C) three times, Extracts were clarified by centrifugation for 10 min at 4°C in a microfuge. Extract (diluted in freeze-thaw buffer to a final volume of 25 ~1) was added to 175 ~1 of assay buffer (60 mM Na,HPO, - 7H,O, 40 mM NaH,PO, 3H,O, 10 mM KCl, 1 mM MgSO, * 7H,O, 50 mM 2-mercaptoethanol, adjusted to pH 7.01, and 40 ~1 of 600 PM 4-methyl umbelliferyl beta-D-galactoside (Sigma>; the assay mixtures were incubated at 30°C for 3 h. Cleavage of the substrate was measured after addition of 100 ~1 1 M Na,CO, using a Fluoroskan I fluorimeter (Flow). Protein concentrations were determined by the procedure of Bradford (1976) using bovine gamma globulin as the standard. Beta-gal activity is presented as fluorescence units per pg of protein.
144
Results Expression of ICP6 We recently constructed a deletion mutant (de122Z) of HSV-l(F) in which the entire coding sequence for the IE protein ICP22 (Vmw68, Usl) was replaced by the ICP6 promoter linked to the bacterial lucZ gene. Complete removal of the ICP22 coding sequence had only minimal effect on the replication of this virus in Vero cells under the conditions used (Poffenberger et al., in press). As part of our characterization of the de122Z mutant, we examined the expression of the ICP6::lucZ fusion protein using a sensitive fluorometric beta-galactosidase assay. Extracts of del22Z-infected Vero cells possessed significant beta-gal activity whereas extracts prepared from uninfected ceils or cells infected with the parental HSV-l(F) had little or no activity when assayed under our standard reaction conditions (data not shown). Regulation of ICP6 expression To examine the regulation of ICP6::lacZ expression by de122Z, we infected the cells under cyclohe~mide reversal conditions (see materials and ~ethods~. Under these conditions only the IE mRNAs and proteins should be synthesized. As shown in Fig. 1, only about 3% of the maximum beta-gal activity was detected per
Extract (~1) Fig. 1. Expression of Inc.7 gene from the ICP6 or ICP4 promoter. Vero cells were infected at an MO1 of 10 in the absence or presence of cycloheximide as outlined in Materials and Methods. Beta-gal activity was measured in the cell extracts using the fluorometric substrate as outlined in Materials and Methods. Symbols: a, cells infected with de122Z; A, cells infected with de1222 under cycloheximide reversal conditions; 0, cells infected with RH105; 0, cells infected with RH105 under cycloheximide reversal conditions.
145
1
2
Extract
3
(pl)
Fig. 2. Expression of 1ucZ from the ICP6 promoter at its normal genomic position. Vero cells were infected as outlined in Materials and Methods and the legend to Fig. 1. Symbols: A, infected with hrR3; A, infected with hrR3 under cycloheximide reversal conditions; 0, infected with RH105; 0, infected with RH105 under cycloheximide reversal conditions. Inset shows data plotted on an expanded scale.
of protein in extracts prepared from the cycloheximide-treated cells when compared to the extracts from untreated cells. As a control, parallel cultures were infected with the HSV-l(KOS) RH105 recombinant which expresses ZucZ under control of the IE ICP4 promoter (Ho and Mocarski, 1988). Essentially identical levels of beta-gal activity were detected in extracts of Vero cells that were infected with RH105 in the absence or presence of cycloheximide (Fig. 1) as would be expected for the expression of an IE gene. This demonstrated that our reversal conditions were indeed capable of allowing translation of the mRNAs that were previously synthesized in the presence of the cycloheximide. The results shown in Fig. 1, therefore, indicated that the ICP6 regulatory sequences functioned primarily as an early promoter when assayed in the context of the de122Z viral genome. Because position may affect the regulation of HSV gene expression, we also examined the expression of beta-gal by the HSV-l(KOS) hrR3 recombinant. This virus has the 1acZ sequence inserted in-frame directly into the ICP6 gene at its normal position within the genome (Goldstein and Weller, 1988). HrR3 expressed only about 3% of the maximal beta-gal activity under cycloheximide reversal conditions, and thus the ICP6 promoter was regulated primarily as an early gene at its authentic position (Fig. 2). The level of beta-gal expressed by hrR3 was significantly and reproducibly higher than that expressed by de122Z, relative to expression by RH105. The higher level of expression may be due to an effect of the position in the genome, to differential activity of the dissimilar ICP6::facZ fusions encoded by these two recombinants, or perhaps to the loss of ICP22 function by de122Z. Nevertheless, although the absolute levels were very different, the relative levels expressed under
pg
146
cycloheximide reversal conditions were unchanged. Therefore, tory mechanisms were unaffected by any of those factors.
the relevant regula-
ICP6 expression in the G5 cell line Yao and Courtney (1989, 1992) have reported that small amounts of the HSV-1 transcription activators ICP4 and ICPO are present in the tegument region of the virion. To rule out potential contributions of virion ICP4 or ICPO to the low level of expression observed under cycloheximide reversal conditions, we also examined the regulation of the ICP6 promoter in our stably transfected Vero clone G5. This cell line carries the identical ICP6::lucZ fusion that was inserted into de122Z, plus the HSV-1 TIF gene under control of the inducible mouse metallothionein promoter (mt-1). Activation of the mt-1 promoter by Zn+2 resulted in the induction of beta-gal activity in a dose- and time-dependent manner in this cell line; northern blot analysis confirmed that the steady-state levels of both the TIF
5
b
Extract
(pl)
Fig. 3. a. Zn +’ induced expression of ICP6::lacZ in G5 cells. TIF synthesis was induced in G5 cells with 120 PM ZnSO, for 18 h. The cells were harvested and extracts assayed for beta-gal activity. Symbols: a, uninduced G5 cells; A, G5 cells induced with Znfz; 0, uninduced Vero cells; 0, Vero cells induced with Znt2. b. Expression of ICP6::lucZ in G5 cells infected with HSV-l(F). Uninduced G5 cells were infected with wildtype HSV-l(F) (MO1 = 10). The cells were harvested and the extracts assayed for beta-gal activity. Symbols: A, G5 cells infected with HSV-l(F); A, G5 cells infected with HSV-l(F) under cycloheximide reversal conditions; 0, mock-infected G5 cells; l, mock-infected G5 cells under cycloheximide reversal conditions. Inset shows data plotted on an expanded scale.
147
and beta-gal mRNAs increased upon induction with the metal ion (data not shown). The beta-gal activity induced by Znf2 in G5 cells increased approximately 5 to 15 times the uninduced level in various experiments (Fig. 3a). These results indicated that the ICP6 promoter was responsive to TIF in the absence of all other HSV-encoded proteins. Infection of G5 cells with HSV
Infection of uninduced G5 cells with wildtype HSV-l(F), in the absence of cycloheximide, yielded a very high level of beta-gal activity, whereas infection under cycloheximide reversal conditions yielded only about 3% of maximal expression (Fig. 3b). Per pg of protein, the amount of beta-gal expressed by infected G5 cells (MO1 of 10) under cycloheximide reversal conditions was approximately equal to that detected when the uninfected cells were induced with Zn+2 (Fig. 3a). Thus, the ICP6 promoter responded identically regardless of its location within the context of the viral or cellular genome. Infection of G5 cells with ICP4 and ICPO mutants
It has been reported that transient transfection of D14 cells [carrying an integrated ICP6 gene(s)] with an ICPO expression plasmid transactivates the ICP6
Extract (~1)
01
23 Extract
(pl)
Fig. 4. Expression of ICP6::IacZ in G5 cells infected with HSV-l(KOS) or IE mutants. Uninduced G5 cells were infected (MO1 = 10) with wildtype HSV-l(KOS), d120 (an ICP4 deletion mutant), or n428 (an ICPO nonsense mutant). The cells were harvested and the extracts assayed for beta-gal activity. Symbols: A, G5 cells infected with HSV-l(KOS); A, G5 cells infected with HSV-l(KOS) under cycloheximide reversal conditions; 0, G5 cells infected with d120; W , G5 cells infected with d120 under cycloheximide reversal conditions; 0, G5 cells infected with n428; 0, G5 cells infected with n428 under cycloheximide reversal conditions. Inset shows data plotted on an,.expanded scale.
148
promoter; transfection with an ICP4 plasmid fails to transactivate ICP6 (Goldstein and Weller, 1988). To identify the IE protein(s) required to transactivate ICP6 and to quantitate the response during a viral infection, we separately infected the G5 cell line (in the absence of added Zn+*) with wildtype HSV-1 or with mutants containing a defect in either the ICP4 or ICPO gene. The results of these experiments are shown in Fig. 4. As was shown above for HSV-l(F), the ICP6 promoter was expressed primarily as an early gene after infection of G5 cells with wildtype HSV-l(KOS). Infection with the HSV-l(KOS) ICP4 deletion mutant d120 gave a pattern of expression that was very similar to that of the wildtype control. Specifically, infection in the absence of cycloheximide gave a high level of beta-gal activity, whereas infection in the presence of cycloheximide yielded only about 1% of that level. However, when the cells were infected with the HSV-l(KOS) ICPO nonsense mutant n428, expression of beta-gal was very low, and similarly low levels were detected in both untreated and cycloheximide-treated cells. The failure to induce high levels of beta-gal activity by n428 indicated that functional ICPO was required to transactivate the ICP6 promoter maximally during viral infection.
Discussion
The data presented here demonstrate that the ICP6 promoter responds weakly to TIF and very strongly to ICPO; it does not respond to ICP4, the major HSV early gene transactivator. Previous reports have shown that ICP6 is transactivated in the presence of cycloheximide (Honess and Roizman, 1974; Watson et al., 1979). Our quantitative analyses demonstrate that this accounts for only about l-3% of the total ICP6 expressed during an infection with viable virus. Because total expression from the ICP6 promoter in its normal position appears to be relatively high (as suggested by expression of fucZ by hrR3), TIF-induced transactivation at only l-3% of the maximum level could represent a significant amount of transcription. We would consequently classify ICP6 as a ‘leaky’ early gene. These results are entirely consistent with those of Zhang and Wagner (1987) who reported that the ICP6 mRNA appears quite rapidly after infection and belongs to a class that they termed ‘early beta’ mRNAs. Expression from the ICP6 promoter in our transfected G5 cell line appears to mirror the expression of this promoter in the infected cell. It is very unlikely that the results obtained with the G5 cells can be explained by the presence of a Zn+*-responsive element in the ICP6 promoter. G5 cells lose the ability to respond to Zn+* after extended passage in culture. However, ICP6::lucZ synthesis can still be induced in such cells by infection with HSV-1; the pattern of expression in the absence and presence of cycloheximide is indistinguishable from that observed with Zn+* -responsive G5 cells (data not shown). Our results support the previously published transient assay data that ICP6 can be transactivated by TIF in the absence of all other viral proteins. However, our results differ from the data for the HSV-2 ICPlO-CAT construct because it is
149
transactivated in Vera cells to about the same level by either TIF or ICPO, albeit at different concentrations of input transactivator DNA (Wymer et al., 1989). This may be indicative of true differences between the ICP6 and ICPlO promoters, or perhaps of differences between transient transfection and viral infection. Persson et al. (1985) previously examined the regulation of ICP6 expression in a cell line that constitutively expresses ICP4. They conclude that ICP4 is sufficient to transactivate ICP6. However, their cell line also constitutively expresses ICP47. In addition, it carries the ICPO promoter and a portion of the ICPO coding sequence. The possibility remains that their cell line synthesizes a truncated form of ICPO that retains sufficient biological activity to transactivate the ICP6 promoter. Furthermore, inactivation of ICPO (as in the n428 mutant) is sufficient to reduce ICP6::ZucZ expression in the G5 cell line to the level obtained under cycloheximide reversal conditions even though the mutant encodes functional ICP4 and ICP47 (Fig. 4). Inspection of the published sequence for HSV-1 strain 17 (McGeoch et al., 1988) reveals a less than perfect match to the TIF consensus c&acting motif S’..TAATGARAT..3’ (S’..AAATGGGAT..3’) that begins 132 nucleotides upstream of the ICP6 initiation site. This sequence overlaps a perfect match to the Ott-1 consensus c&-acting site S’..ATGCAAAT..3’ (i.e., S’..ATGCAAATGGGATAC..3’). Since expression of beta-gal in the transfected cell line is dependent upon the Zn+2 -induced synthesis of TIF, binding of Ott-1 to its consensus c&site may be insufficient for transactivation of the ICP6 promoter. The relatively low level of expression (compared to that induced by ICPO) suggests that the imperfect TIF &-acting site may be bound only inefficiently. Other deviations from the consensus GARAT sequence that reduce transactivation by TIF at the IE ICP27 promoter have been described recently (Spector et al., 1991). The ICP4 d120 deletion mutant induces beta-gal in G5 cells to almost the same level as the wildtype virus. This implies that ICPO does not simply stimulate the synthesis or function of ICP4 which, in turn, transactivates the ICP6 promoter. Furthermore, the ICPO amber mutant n428 is unable to induce high levels of beta-gal activity even at an MO1 of 10, a condition where the mutant phenotype is no longer obvious (Cai and Schaffer, 1992). Transient transfections of the G5 cell line (in the absence of added Znf2) with plasmids expressing mutant forms of TIF or ICPO, in conjunction with site-specific mutagenesis of the ICP6 promoter and DNA footprinting analyses, should help to elucidate the requirements for these transactivation events. The gene for RR1 from both HSV-1 (ICP6; Ali et al., 1991) and HSV-2 (ICPlO; Chung et al., 1989; Ali et al., 1991) encodes a protein kinase domain near the 5’ terminus that is apparently not essential for reductase activity. The kinase phosphorylates itself (Ali et al., 1991) but other viral and/or cellular targets may exist; among the IE proteins ICP4, ICPO and ICP22 are all phosphorylated (Pereira et al., 1977; Marsden et al., 1978; Lemaster and Roizman, 1979; Hay and Hay, 1980; Wilcox et al., 1980). It is possible that low level transactivation of RR1 by TIF may be required for the rapid production of the kinase at IE times postinfection. Subsequent high level transactivation by ICPO would provide adequate RR1 to
150
assemble with the smaller RR2 subunit (HSV-1 UL40) into active ribonucleotide reductase. Studies of HSV latency suggest that ICPO may be one of the principal components required for reactivation (Russell et al., 1987; Leib et al., 1989). Consequently, expression of ICPO at the onset of reactivation is likely to have a direct effect on the expression of ICP6. This could provide a mechanism for the synthesis of a potentially important kinase in the absence of TIF (which is expressed as a late protein) or ICP4 (which does not activate the ICP6 promoter). Thus, the dual regulation of the ICP6 promoter by TIF and ICPO could provide RRl/kinase expression at the appropriate time under very different physiological conditions (i.e., lytic infection vs. reactivation). Experiments are in progress to test some of the predictions made by this hypothesis.
Acknowledgements
We thank E. Mocarski, B. Roizman, P. Schaffer and S. Weller for providing biological reagents, M. Levine and R. Jariwalla for providing access to data prior to publication, and M. Levine, R. Jariwalla, R. Sandri-Goldin, E. Wagner and S. Weller for informative discussions and comments on this manuscript.
References Ali, M.A., McWeeney, D., Milosavljevic, A., Jurka, J. and Jariwalla, R.J. (1991) Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2. Proc. Natl. Acad. Sci. USA 88, 8257-8261. Bradford, M.A. (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Cai, W. and Schaffer, P.A. (1989) Herpes simplex virus type 1 ICPO plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA.J. Virol. 63, 4579-4589. Cai, W. and Schaffer, P.A. (1991) A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICPO, a transactivating protein of herpes simplex virus type 1. J. Viral. 65, 4078-4090. Chung, T.D., Wymer, J.P., Smith, C.C., Kulka, M. and Aurelian, L. (1989) Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICPlO). J. Virol. 63, 3389-3398. Cameron, J.M., McDougall, I., Marsden, H.S., Preston, V.G., Ryan, D.M. and Subak-Sharpe, J.H. (1988) Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target. J. Gen. Virol. 69, 2607-2612. DeLuca, N.A., McCarthy, A.M. and Schaffer, P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J. Virol. 56, 558-570. Gerster, T. and Roeder, R.G. (1988) A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc. Natl. Acad. Sci. USA 85, 6347-6351. Goldstein, D.J. and Weller S.K. (1988a) Herpes simplex virus type l-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 1ucZ insertion mutant. J. Virol. 62, 196-205.
1.51 Goldstein, D.J. and Weller S.K. (1988b) An ICP6::lucZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J. Virol. 62, 2970-2977. Greaves, R. and O’Hare, P. (1989) Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65. J. Virol. 63, 1641-1650. Greaves, R. and O’Hare, P. (1990) Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences. J. Virol. 64, 2716-2724. Haigh, A., Greaves, R. and O’Hare, P. (1990) Interference with the assembly of a virus-host transcription complex by peptide competition. Nature 344, 257-259. Hay, R.T. and Hay, J. (1980) Properties of herpes virus-induced ‘immediate early’ polypeptides. Virology 104, 230-2345. Ho, D.Y. and Mocarski, ES. (1988) Beta-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse. Virology 167, 279-283. Honess, R.W. and Roizman, B. (1974) Regulation of herpes virus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 8-19. Honess, R.W. and Roizman, B. (1975) Regulation of herpes virus macromolecular synthesis: sequential transcription of polypeptide synthesis requires functional viral polypeptides. Proc. Natl. Acad. Sci. USA 72, 1276-1280. Idowu, A.D., Fraser-Smith, E.B., Poffenberger, K.L. and Herman, R.C. (1992) Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in Go. Antiviral Res. 17, 145-156. Jacobson, J.G., Leib, D.A., Goldstein D.J., Bogard, CL., Schaffer, P.A., Weller, S.K. and Coen, D.M. (1989) A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells. Virology 173, 276-283. Kristie, T.M., LeBowitz, J.H. and Sharp, P.A. (1989) The octamer-binding proteins form multi-proteinDNA complexes with the HSV alpha-TIF regulatory protein. EMBO J. 8, 4229-4238. Kristie, T.M. and Roizman, B. (1987) Host cell proteins bind to the c&-acting site required for virion-mediated induction of herpes simplex virus 1 alpha genes. Proc. Natl. Acad. Sci. USA 84, 71-75. Kristie, T.M. and Roizman, B. (1988) Differentiation and DNA contact points of host proteins binding at the cis site for virion-mediated induction of alpha genes of herpes simplex virus 1. J. Virol. 62, 1145-1157. Kristie, T.M. and Sharp, P.A. (1990) Interactions of the Ott-1 POU subdomains with specific DNA sequences and with the HSV alpha-trans-activator protein. Genes Dev. 4, 2383-2396. Leib, D.A., Coen, D.M., Bogard, C.L., Hicks, K.A., Yager, D. R., Knipe, D.M., Tyler, K.L. and Schaffer, P.A. (1989) Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J. Virol. 63, 759-768. Lemaster, S. and Roizman, B. (1979) Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. J. Virol. 3.5, 798-811. Mackem, S. and Roizman, B. (1982) Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes. J. Virol. 44, 939-949. Marsden, H.S., Stow, N.D., Preston, V.G., Timbury, M.C. and Wilkie, N.M. (1978) Physical mapping of herpes simplex virus induced polypeptides. J. Virol. 28, 624-642. McGeoch, D.J., Dalrymple, M.A., Davison, A.J., Dolan, A., Frame, M.C., McNab, D., Perry, L.J., Scott, J.E. and Taylor, P. (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69, 1531-1574. M&night, J.L.C., Kristie, T.M. and Roizman, B. (1987) Binding of the virion protein mediating alpha gene induction in herpes simplex virus l-infected cells to its &-site requires cellular proteins. Proc. Natl. Acad. Sci. USA 84, 7061-7065.
152 O’Hare, P. and Goding, C.R. (1988) Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell 52. 435-445. O’Hare, P., Coding, CR. and Haigh, A. (1988) Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression. EMBO J. 7, 4231-4238. O’Neill, E.A., Fletcher, C., Burrow, C.R., Heintz, N., Roeder, R.G. and Kelly, T.G. (1988) Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III. Science 241, 1210-1213. Pereira, L., Wolff, M.H., Fenwick, M. and Roizman, B. (19771 Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells. Virology 71, 733-749. Persson, R.H., Bacchetti, S. and Smiley, J.R. (1985) Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans. J. Virol. 54, 414-421. Poffenberger. K.L., Raichlen, P.E. and Herman, R.C. In vitro characterization of a herpes simplex virus type 1 ICP22 null mutant. Virus Genes, in press. Preston, CM., Frame, M.C. and Campbell, M.E.M. (1988) A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence. Cell 52, 425-434. Russell, .I., Stow, N.D., Stow, EC. and Preston, CM. (1987) Herpes simplex virus genes involved in latency in c&u. J. Gen. Viral. 68, 3~9-3~18. Spaete, R.R. and Mocarski, E.S. (1985) Regulation of cytomegalovirus gene expression: alpha and beta promoters are transactivated by viral functions in permissive human fibroblasts. J. Viral. 56, 135-143. Spector, D., Purves, F. and Roizman, B. (1990) Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome. Proc. Nat]. Acad. Sci. USA 87. 5268-5272. Spector, D., Purves, F. and Roizman, B. (1991) Roie of alpha-transducing factor (VP16) in the induction of alpha genes within the context of viral genomes. J. Viral. 65, 3504-3513. Stern, S., Tanaka, M. and Herr, W. (1989) The act-1 homeodomain directs formation of a multiproteinDNA complex with HSV transactivator VP16. Nature 341, 624-630. Sturm, R., Das, G. and Herr, W. (1988) The ubiquitous octamer binding act-1 contains a POU domain with a homeobox subdomain. Genes Dev. 2, 1582-1599. Triezenberg, S.J., Kingsbury, R.C. and M&night, S.L. (1988) Functional dissection of VP16 the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718-729. Turk, S.R., Kik, N.A., Birch, G.M., Cbiego Jr., D.J. and Shipman Jr., C. (1989) Herpes simplex virus type-l ribonucleotide reductase null mutants induce lesions in guinea pigs. Virology 173, 733-735. Wilcox, K.W., Kohn, A., Sklyanskaya, E. and Roizman, B. (1980) Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA.J. Virol. 33, 167-182. Watson, R.J., Preston, CM., and Clements, J.B. (1979) Separation and characterization of herpes simplex virus type 1 immediate early mRNAs. J. Viroi. 31, 42-52. Wymer, J.P., Chung, T.D., Chang, Y.N., Hayward, GS. and Aurelian, L. (1989) Identification of immediate-early-type cis-response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2. J. Virol. 63, 2773-2784. Xiao, P. and Capone, J.P. (1990) A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with act-1. Mol. Cetl. Biol. 10, 4974-4977. Yao, F. and Courtney, R.J. (1989) A major transcriptional regulatory protein (ICP41 of herpes simplex virus type 1 is associated with purified virions. J. Viral. 63, 3338-3344. Yao, F. and Courtney, R.J. (1992) Association of ICPO but not ICP27 with purified virions of herpes simplex virus type 1. Zhang, Y.F. and Wagner, E.K. (1987) The kinetics of expression of individual herpes simplex virus type 1 transcripts. Virus Genes 1, 49-60.
VIRUS
141 B.V. All rights reserved
016%1702/92/$05.00
00834
The herpes simplex virus type 1 ICP6 gene is regulated by a ‘leaky’ early promoter * Ping Sze and Ronald
C. Herman
Syntev Research, Palo Alto, CA 94304, USA (Received
18 May 1992; revision
received
and accepted
3 August
1992)
Summary
Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the ZucZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICPO as the major transactivator of the ICP6 promoter. Herpes simplex virus; Gene expression; ICP6; ICPO
Introduction
Three classes of herpes simplex virus (HSV) genes have been recognized on the basis of the timing and regulation of their expression after infection (Honess and Roizman, 1974, 1975). The five immediate early (IE) (or alpha) genes are expressed immediately after infection and are transcribed even in the presence of a high concentration of the protein synthesis inhibitor cycloheximide. Expression of Correspondence to: R.C. Herman, Syntex Research, 3401 Hillview Avenue, Fax: (1) (415) 354-7363, after 28 December 1992: (1) (415) 796-7363. * Manuscript no. 351 from the Institute of Bio-Organic Chemistry.
Palo Alto, CA 94304, USA.
142
the IE genes is dependent upon the interaction of the virion tegument protein known as the alpha-transinducing factor (TIF; alternatively called Vmw65, VP16, ICP25, or UL48) with the POU domain cellular transcription factor Ott-1 (Kristie and Roizman, 1987, 1988; O’Hare and Goding, 1988; O’Neill et al., 1988; Sturm et al., 1988; Kristie and Sharp, 1990) and other cellular proteins (M&night et al., 1987; Gerster and Roeder, 1988; O’Hare et al., 1988; Preston et al., 1988; Triezenberg et al., 1988; Greaves and O’Hare, 1989, 1990; Kristie et al., 1989; Stern et al., 1989; Haigh et al., 1990; Xiao and Capone, 1990). Genes responsive to transactivation by TIF have 5’. . . TAATGARAT.. .3’ or related consensus sequence (Mackem and Roizman, 1982; Spector et al., 1990, 19911, or 5’. . . GCGGAA.. .3’ consensus sequence (Triezenberg, 1988) in the upstream regulatory region. Early (beta) genes require the presence of one or more of the IE proteins for normal expression. Late (gamma) genes are expressed after the onset of DNA replication, and can be further subdivided into a group that requires DNA synthesis for expression and a group that does not. ICP6 (UL39) encodes the large subunit (RRl) of the HSV-1 ribonucleotide reductase that is important for viral DNA synthesis as well as for neurovirulence and reactivation in several animal models (Cameron et al., 1988; Jacobson et al., 1989; Turk et al., 1989; Idowu et al., 1992). It has been suggested that ICP6 is an IE gene because its mRNA can be detected after infection in the presence of cycloheximide (Honess and Roizman, 1974; Watson et al., 1979). Others have shown that ICP6 and the HSV-2 equivalent ICPlO are responsive to TIF in transient expression assays perhaps because of the presence of an upstream TAATGARAT-like element. Transient assays also suggest that the promoter is responsive to ICPO in some cell lines but it is apparently not responsive to ICP4 (Goldstein and Weller, 1988b; Wymer et al., 1989). We report here the quantitative results of experiments designed to examine the regulation of the ICP6 gene in infected Vero cells and in a stably transfected cell line. These results indicate that the ICP6 promoter is weakly transactivated by TIF and maximally induced by ICPO, but is not activated by ICP4.
Materials
and Methods
Cells and Liruses
Vero cells were grown in minimal essential medium supplemented with 5% fetal calf serum and non-essential amino acids. The G5 cell line is a stably transfected Vero clone that carries the HSV-1 TIF gene under control of the inducible mouse metallothionein (mt-1) promoter (plasmid pRB3522 obtained from B. Roizman) plus the same ICP6::lacZ fusion that was inserted into the HSV-1 de122Z mutant (see below); the cells were grown in media supplemented as above plus G418 (500 pgg/ml; GIBCO). Where indicated, expression of TIF was induced by incubating the cells for 18 h in media supplemented as above plus 120 PM ZnSO,. De122Z is a deletion mutant of HSV-l(F) in which the coding sequence for ICP22 (Usl) was replaced by the ICP6 promoter linked to the bacterial 1acZ gene.
143
In this const~ction, 1.19 kb of upstream sequence, including the codons for the first 60 amino acids of ICP6, were fused in frame to 1ucZ (Goldstein and Weller, 1988b). A detailed description of the de122Z mutant will be presented elsewhere (Poffenberger et al., in press). HSV-l(KOS) RH105 is a recombinant virus (obtained from E. Mocarski) that carries the E. co/i 1ucZ gene under the control of the ICP4 promoter and inserted into the HSV thymidine kinase gene (Ho and Mocarski, 1988). HSV-l(KOS) hrR3 is a recombinant virus (obtained from S. Weller) that has the la& gene inserted in-frame directly into the ICP6 RR1 coding sequence following the codon for amino acid 434 (via the BarnHI site at map coordinate 0.574) (Goldstein and Weller, 1988a). HSV-l(KOS) n428 is a nonsense mutant (obtained from P. Schaffer) that contains a synthetic nonsense N&I linker inserted into the ICPO coding sequence and disrupting the reading frame at amino acid 428 (Cai and Sehaffer, 1989). HSV-l(KOS) d120 is a deletion mutant (obtained from P. Schaffer) from which the coding sequence for ICP4 was deleted (DeLuca et al., 1985). Infections and cycloheximide reversal conditions
The cells were infected in the absence or presence of ~cloheximide (100 fig/ml) at a multipli~i~ of infection (MOI) of 10. Virus was adsorbed for 1 h at 37°C; the inoculum was removed and replaced with fresh warm media, and the cells were incubated at 37°C. Extracts were prepared from cells infected in the absence of cycloheximide at 6 h postinfection (p.i.>. When cycloheximide was used, the cells were pretreated for 30 min and then infected in the presence of the drug. The media containing ~ycloheximide was removed at 6 h p.i.; the cells were washed three times with media containing actinomycin D (5 pg/ml). The infection was allowed to proceed in the presence of actinomycin D. Extracts of cells infected under the reversal conditions were prepared 4 h after removal of the cycloheximide (10 h p.i.). zeta-ga~actosidase ~~-ga~~assay
Beta-gal activity was determined essentially as previously described (Spaetc and Mocarski, 1985). Cells in lOO-mm dishes were washed with cold phosphate-buffered saline (PBS), scraped into 1 ml of PBS, and suspended in 5 ml PBS. Cells were pelleted, resuspended in 100 ~1 of freeze-thaw buffer (0.25 M sucrose, 10 mM Tris-HCI, pH 7.4, 10 mM EDTA) and alternately frozen (-SO*C> and thawed (37°C) three times, Extracts were clarified by centrifugation for 10 min at 4°C in a microfuge. Extract (diluted in freeze-thaw buffer to a final volume of 25 ~1) was added to 175 ~1 of assay buffer (60 mM Na,HPO, - 7H,O, 40 mM NaH,PO, 3H,O, 10 mM KCl, 1 mM MgSO, * 7H,O, 50 mM 2-mercaptoethanol, adjusted to pH 7.01, and 40 ~1 of 600 PM 4-methyl umbelliferyl beta-D-galactoside (Sigma>; the assay mixtures were incubated at 30°C for 3 h. Cleavage of the substrate was measured after addition of 100 ~1 1 M Na,CO, using a Fluoroskan I fluorimeter (Flow). Protein concentrations were determined by the procedure of Bradford (1976) using bovine gamma globulin as the standard. Beta-gal activity is presented as fluorescence units per pg of protein.
144
Results Expression of ICP6 We recently constructed a deletion mutant (de122Z) of HSV-l(F) in which the entire coding sequence for the IE protein ICP22 (Vmw68, Usl) was replaced by the ICP6 promoter linked to the bacterial lucZ gene. Complete removal of the ICP22 coding sequence had only minimal effect on the replication of this virus in Vero cells under the conditions used (Poffenberger et al., in press). As part of our characterization of the de122Z mutant, we examined the expression of the ICP6::lucZ fusion protein using a sensitive fluorometric beta-galactosidase assay. Extracts of del22Z-infected Vero cells possessed significant beta-gal activity whereas extracts prepared from uninfected ceils or cells infected with the parental HSV-l(F) had little or no activity when assayed under our standard reaction conditions (data not shown). Regulation of ICP6 expression To examine the regulation of ICP6::lacZ expression by de122Z, we infected the cells under cyclohe~mide reversal conditions (see materials and ~ethods~. Under these conditions only the IE mRNAs and proteins should be synthesized. As shown in Fig. 1, only about 3% of the maximum beta-gal activity was detected per
Extract (~1) Fig. 1. Expression of Inc.7 gene from the ICP6 or ICP4 promoter. Vero cells were infected at an MO1 of 10 in the absence or presence of cycloheximide as outlined in Materials and Methods. Beta-gal activity was measured in the cell extracts using the fluorometric substrate as outlined in Materials and Methods. Symbols: a, cells infected with de122Z; A, cells infected with de1222 under cycloheximide reversal conditions; 0, cells infected with RH105; 0, cells infected with RH105 under cycloheximide reversal conditions.
145
1
2
Extract
3
(pl)
Fig. 2. Expression of 1ucZ from the ICP6 promoter at its normal genomic position. Vero cells were infected as outlined in Materials and Methods and the legend to Fig. 1. Symbols: A, infected with hrR3; A, infected with hrR3 under cycloheximide reversal conditions; 0, infected with RH105; 0, infected with RH105 under cycloheximide reversal conditions. Inset shows data plotted on an expanded scale.
of protein in extracts prepared from the cycloheximide-treated cells when compared to the extracts from untreated cells. As a control, parallel cultures were infected with the HSV-l(KOS) RH105 recombinant which expresses ZucZ under control of the IE ICP4 promoter (Ho and Mocarski, 1988). Essentially identical levels of beta-gal activity were detected in extracts of Vero cells that were infected with RH105 in the absence or presence of cycloheximide (Fig. 1) as would be expected for the expression of an IE gene. This demonstrated that our reversal conditions were indeed capable of allowing translation of the mRNAs that were previously synthesized in the presence of the cycloheximide. The results shown in Fig. 1, therefore, indicated that the ICP6 regulatory sequences functioned primarily as an early promoter when assayed in the context of the de122Z viral genome. Because position may affect the regulation of HSV gene expression, we also examined the expression of beta-gal by the HSV-l(KOS) hrR3 recombinant. This virus has the 1acZ sequence inserted in-frame directly into the ICP6 gene at its normal position within the genome (Goldstein and Weller, 1988). HrR3 expressed only about 3% of the maximal beta-gal activity under cycloheximide reversal conditions, and thus the ICP6 promoter was regulated primarily as an early gene at its authentic position (Fig. 2). The level of beta-gal expressed by hrR3 was significantly and reproducibly higher than that expressed by de122Z, relative to expression by RH105. The higher level of expression may be due to an effect of the position in the genome, to differential activity of the dissimilar ICP6::facZ fusions encoded by these two recombinants, or perhaps to the loss of ICP22 function by de122Z. Nevertheless, although the absolute levels were very different, the relative levels expressed under
pg
146
cycloheximide reversal conditions were unchanged. Therefore, tory mechanisms were unaffected by any of those factors.
the relevant regula-
ICP6 expression in the G5 cell line Yao and Courtney (1989, 1992) have reported that small amounts of the HSV-1 transcription activators ICP4 and ICPO are present in the tegument region of the virion. To rule out potential contributions of virion ICP4 or ICPO to the low level of expression observed under cycloheximide reversal conditions, we also examined the regulation of the ICP6 promoter in our stably transfected Vero clone G5. This cell line carries the identical ICP6::lucZ fusion that was inserted into de122Z, plus the HSV-1 TIF gene under control of the inducible mouse metallothionein promoter (mt-1). Activation of the mt-1 promoter by Zn+2 resulted in the induction of beta-gal activity in a dose- and time-dependent manner in this cell line; northern blot analysis confirmed that the steady-state levels of both the TIF
5
b
Extract
(pl)
Fig. 3. a. Zn +’ induced expression of ICP6::lacZ in G5 cells. TIF synthesis was induced in G5 cells with 120 PM ZnSO, for 18 h. The cells were harvested and extracts assayed for beta-gal activity. Symbols: a, uninduced G5 cells; A, G5 cells induced with Znfz; 0, uninduced Vero cells; 0, Vero cells induced with Znt2. b. Expression of ICP6::lucZ in G5 cells infected with HSV-l(F). Uninduced G5 cells were infected with wildtype HSV-l(F) (MO1 = 10). The cells were harvested and the extracts assayed for beta-gal activity. Symbols: A, G5 cells infected with HSV-l(F); A, G5 cells infected with HSV-l(F) under cycloheximide reversal conditions; 0, mock-infected G5 cells; l, mock-infected G5 cells under cycloheximide reversal conditions. Inset shows data plotted on an expanded scale.
147
and beta-gal mRNAs increased upon induction with the metal ion (data not shown). The beta-gal activity induced by Znf2 in G5 cells increased approximately 5 to 15 times the uninduced level in various experiments (Fig. 3a). These results indicated that the ICP6 promoter was responsive to TIF in the absence of all other HSV-encoded proteins. Infection of G5 cells with HSV
Infection of uninduced G5 cells with wildtype HSV-l(F), in the absence of cycloheximide, yielded a very high level of beta-gal activity, whereas infection under cycloheximide reversal conditions yielded only about 3% of maximal expression (Fig. 3b). Per pg of protein, the amount of beta-gal expressed by infected G5 cells (MO1 of 10) under cycloheximide reversal conditions was approximately equal to that detected when the uninfected cells were induced with Zn+2 (Fig. 3a). Thus, the ICP6 promoter responded identically regardless of its location within the context of the viral or cellular genome. Infection of G5 cells with ICP4 and ICPO mutants
It has been reported that transient transfection of D14 cells [carrying an integrated ICP6 gene(s)] with an ICPO expression plasmid transactivates the ICP6
Extract (~1)
01
23 Extract
(pl)
Fig. 4. Expression of ICP6::IacZ in G5 cells infected with HSV-l(KOS) or IE mutants. Uninduced G5 cells were infected (MO1 = 10) with wildtype HSV-l(KOS), d120 (an ICP4 deletion mutant), or n428 (an ICPO nonsense mutant). The cells were harvested and the extracts assayed for beta-gal activity. Symbols: A, G5 cells infected with HSV-l(KOS); A, G5 cells infected with HSV-l(KOS) under cycloheximide reversal conditions; 0, G5 cells infected with d120; W , G5 cells infected with d120 under cycloheximide reversal conditions; 0, G5 cells infected with n428; 0, G5 cells infected with n428 under cycloheximide reversal conditions. Inset shows data plotted on an,.expanded scale.
148
promoter; transfection with an ICP4 plasmid fails to transactivate ICP6 (Goldstein and Weller, 1988). To identify the IE protein(s) required to transactivate ICP6 and to quantitate the response during a viral infection, we separately infected the G5 cell line (in the absence of added Zn+*) with wildtype HSV-1 or with mutants containing a defect in either the ICP4 or ICPO gene. The results of these experiments are shown in Fig. 4. As was shown above for HSV-l(F), the ICP6 promoter was expressed primarily as an early gene after infection of G5 cells with wildtype HSV-l(KOS). Infection with the HSV-l(KOS) ICP4 deletion mutant d120 gave a pattern of expression that was very similar to that of the wildtype control. Specifically, infection in the absence of cycloheximide gave a high level of beta-gal activity, whereas infection in the presence of cycloheximide yielded only about 1% of that level. However, when the cells were infected with the HSV-l(KOS) ICPO nonsense mutant n428, expression of beta-gal was very low, and similarly low levels were detected in both untreated and cycloheximide-treated cells. The failure to induce high levels of beta-gal activity by n428 indicated that functional ICPO was required to transactivate the ICP6 promoter maximally during viral infection.
Discussion
The data presented here demonstrate that the ICP6 promoter responds weakly to TIF and very strongly to ICPO; it does not respond to ICP4, the major HSV early gene transactivator. Previous reports have shown that ICP6 is transactivated in the presence of cycloheximide (Honess and Roizman, 1974; Watson et al., 1979). Our quantitative analyses demonstrate that this accounts for only about l-3% of the total ICP6 expressed during an infection with viable virus. Because total expression from the ICP6 promoter in its normal position appears to be relatively high (as suggested by expression of fucZ by hrR3), TIF-induced transactivation at only l-3% of the maximum level could represent a significant amount of transcription. We would consequently classify ICP6 as a ‘leaky’ early gene. These results are entirely consistent with those of Zhang and Wagner (1987) who reported that the ICP6 mRNA appears quite rapidly after infection and belongs to a class that they termed ‘early beta’ mRNAs. Expression from the ICP6 promoter in our transfected G5 cell line appears to mirror the expression of this promoter in the infected cell. It is very unlikely that the results obtained with the G5 cells can be explained by the presence of a Zn+*-responsive element in the ICP6 promoter. G5 cells lose the ability to respond to Zn+* after extended passage in culture. However, ICP6::lucZ synthesis can still be induced in such cells by infection with HSV-1; the pattern of expression in the absence and presence of cycloheximide is indistinguishable from that observed with Zn+* -responsive G5 cells (data not shown). Our results support the previously published transient assay data that ICP6 can be transactivated by TIF in the absence of all other viral proteins. However, our results differ from the data for the HSV-2 ICPlO-CAT construct because it is
149
transactivated in Vera cells to about the same level by either TIF or ICPO, albeit at different concentrations of input transactivator DNA (Wymer et al., 1989). This may be indicative of true differences between the ICP6 and ICPlO promoters, or perhaps of differences between transient transfection and viral infection. Persson et al. (1985) previously examined the regulation of ICP6 expression in a cell line that constitutively expresses ICP4. They conclude that ICP4 is sufficient to transactivate ICP6. However, their cell line also constitutively expresses ICP47. In addition, it carries the ICPO promoter and a portion of the ICPO coding sequence. The possibility remains that their cell line synthesizes a truncated form of ICPO that retains sufficient biological activity to transactivate the ICP6 promoter. Furthermore, inactivation of ICPO (as in the n428 mutant) is sufficient to reduce ICP6::ZucZ expression in the G5 cell line to the level obtained under cycloheximide reversal conditions even though the mutant encodes functional ICP4 and ICP47 (Fig. 4). Inspection of the published sequence for HSV-1 strain 17 (McGeoch et al., 1988) reveals a less than perfect match to the TIF consensus c&acting motif S’..TAATGARAT..3’ (S’..AAATGGGAT..3’) that begins 132 nucleotides upstream of the ICP6 initiation site. This sequence overlaps a perfect match to the Ott-1 consensus c&-acting site S’..ATGCAAAT..3’ (i.e., S’..ATGCAAATGGGATAC..3’). Since expression of beta-gal in the transfected cell line is dependent upon the Zn+2 -induced synthesis of TIF, binding of Ott-1 to its consensus c&site may be insufficient for transactivation of the ICP6 promoter. The relatively low level of expression (compared to that induced by ICPO) suggests that the imperfect TIF &-acting site may be bound only inefficiently. Other deviations from the consensus GARAT sequence that reduce transactivation by TIF at the IE ICP27 promoter have been described recently (Spector et al., 1991). The ICP4 d120 deletion mutant induces beta-gal in G5 cells to almost the same level as the wildtype virus. This implies that ICPO does not simply stimulate the synthesis or function of ICP4 which, in turn, transactivates the ICP6 promoter. Furthermore, the ICPO amber mutant n428 is unable to induce high levels of beta-gal activity even at an MO1 of 10, a condition where the mutant phenotype is no longer obvious (Cai and Schaffer, 1992). Transient transfections of the G5 cell line (in the absence of added Znf2) with plasmids expressing mutant forms of TIF or ICPO, in conjunction with site-specific mutagenesis of the ICP6 promoter and DNA footprinting analyses, should help to elucidate the requirements for these transactivation events. The gene for RR1 from both HSV-1 (ICP6; Ali et al., 1991) and HSV-2 (ICPlO; Chung et al., 1989; Ali et al., 1991) encodes a protein kinase domain near the 5’ terminus that is apparently not essential for reductase activity. The kinase phosphorylates itself (Ali et al., 1991) but other viral and/or cellular targets may exist; among the IE proteins ICP4, ICPO and ICP22 are all phosphorylated (Pereira et al., 1977; Marsden et al., 1978; Lemaster and Roizman, 1979; Hay and Hay, 1980; Wilcox et al., 1980). It is possible that low level transactivation of RR1 by TIF may be required for the rapid production of the kinase at IE times postinfection. Subsequent high level transactivation by ICPO would provide adequate RR1 to
150
assemble with the smaller RR2 subunit (HSV-1 UL40) into active ribonucleotide reductase. Studies of HSV latency suggest that ICPO may be one of the principal components required for reactivation (Russell et al., 1987; Leib et al., 1989). Consequently, expression of ICPO at the onset of reactivation is likely to have a direct effect on the expression of ICP6. This could provide a mechanism for the synthesis of a potentially important kinase in the absence of TIF (which is expressed as a late protein) or ICP4 (which does not activate the ICP6 promoter). Thus, the dual regulation of the ICP6 promoter by TIF and ICPO could provide RRl/kinase expression at the appropriate time under very different physiological conditions (i.e., lytic infection vs. reactivation). Experiments are in progress to test some of the predictions made by this hypothesis.
Acknowledgements
We thank E. Mocarski, B. Roizman, P. Schaffer and S. Weller for providing biological reagents, M. Levine and R. Jariwalla for providing access to data prior to publication, and M. Levine, R. Jariwalla, R. Sandri-Goldin, E. Wagner and S. Weller for informative discussions and comments on this manuscript.
References Ali, M.A., McWeeney, D., Milosavljevic, A., Jurka, J. and Jariwalla, R.J. (1991) Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2. Proc. Natl. Acad. Sci. USA 88, 8257-8261. Bradford, M.A. (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Cai, W. and Schaffer, P.A. (1989) Herpes simplex virus type 1 ICPO plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA.J. Virol. 63, 4579-4589. Cai, W. and Schaffer, P.A. (1991) A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICPO, a transactivating protein of herpes simplex virus type 1. J. Viral. 65, 4078-4090. Chung, T.D., Wymer, J.P., Smith, C.C., Kulka, M. and Aurelian, L. (1989) Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICPlO). J. Virol. 63, 3389-3398. Cameron, J.M., McDougall, I., Marsden, H.S., Preston, V.G., Ryan, D.M. and Subak-Sharpe, J.H. (1988) Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target. J. Gen. Virol. 69, 2607-2612. DeLuca, N.A., McCarthy, A.M. and Schaffer, P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J. Virol. 56, 558-570. Gerster, T. and Roeder, R.G. (1988) A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc. Natl. Acad. Sci. USA 85, 6347-6351. Goldstein, D.J. and Weller S.K. (1988a) Herpes simplex virus type l-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 1ucZ insertion mutant. J. Virol. 62, 196-205.
1.51 Goldstein, D.J. and Weller S.K. (1988b) An ICP6::lucZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J. Virol. 62, 2970-2977. Greaves, R. and O’Hare, P. (1989) Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65. J. Virol. 63, 1641-1650. Greaves, R. and O’Hare, P. (1990) Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences. J. Virol. 64, 2716-2724. Haigh, A., Greaves, R. and O’Hare, P. (1990) Interference with the assembly of a virus-host transcription complex by peptide competition. Nature 344, 257-259. Hay, R.T. and Hay, J. (1980) Properties of herpes virus-induced ‘immediate early’ polypeptides. Virology 104, 230-2345. Ho, D.Y. and Mocarski, ES. (1988) Beta-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse. Virology 167, 279-283. Honess, R.W. and Roizman, B. (1974) Regulation of herpes virus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 8-19. Honess, R.W. and Roizman, B. (1975) Regulation of herpes virus macromolecular synthesis: sequential transcription of polypeptide synthesis requires functional viral polypeptides. Proc. Natl. Acad. Sci. USA 72, 1276-1280. Idowu, A.D., Fraser-Smith, E.B., Poffenberger, K.L. and Herman, R.C. (1992) Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in Go. Antiviral Res. 17, 145-156. Jacobson, J.G., Leib, D.A., Goldstein D.J., Bogard, CL., Schaffer, P.A., Weller, S.K. and Coen, D.M. (1989) A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells. Virology 173, 276-283. Kristie, T.M., LeBowitz, J.H. and Sharp, P.A. (1989) The octamer-binding proteins form multi-proteinDNA complexes with the HSV alpha-TIF regulatory protein. EMBO J. 8, 4229-4238. Kristie, T.M. and Roizman, B. (1987) Host cell proteins bind to the c&-acting site required for virion-mediated induction of herpes simplex virus 1 alpha genes. Proc. Natl. Acad. Sci. USA 84, 71-75. Kristie, T.M. and Roizman, B. (1988) Differentiation and DNA contact points of host proteins binding at the cis site for virion-mediated induction of alpha genes of herpes simplex virus 1. J. Virol. 62, 1145-1157. Kristie, T.M. and Sharp, P.A. (1990) Interactions of the Ott-1 POU subdomains with specific DNA sequences and with the HSV alpha-trans-activator protein. Genes Dev. 4, 2383-2396. Leib, D.A., Coen, D.M., Bogard, C.L., Hicks, K.A., Yager, D. R., Knipe, D.M., Tyler, K.L. and Schaffer, P.A. (1989) Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J. Virol. 63, 759-768. Lemaster, S. and Roizman, B. (1979) Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. J. Virol. 3.5, 798-811. Mackem, S. and Roizman, B. (1982) Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes. J. Virol. 44, 939-949. Marsden, H.S., Stow, N.D., Preston, V.G., Timbury, M.C. and Wilkie, N.M. (1978) Physical mapping of herpes simplex virus induced polypeptides. J. Virol. 28, 624-642. McGeoch, D.J., Dalrymple, M.A., Davison, A.J., Dolan, A., Frame, M.C., McNab, D., Perry, L.J., Scott, J.E. and Taylor, P. (1988) The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69, 1531-1574. M&night, J.L.C., Kristie, T.M. and Roizman, B. (1987) Binding of the virion protein mediating alpha gene induction in herpes simplex virus l-infected cells to its &-site requires cellular proteins. Proc. Natl. Acad. Sci. USA 84, 7061-7065.
152 O’Hare, P. and Goding, C.R. (1988) Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell 52. 435-445. O’Hare, P., Coding, CR. and Haigh, A. (1988) Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression. EMBO J. 7, 4231-4238. O’Neill, E.A., Fletcher, C., Burrow, C.R., Heintz, N., Roeder, R.G. and Kelly, T.G. (1988) Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III. Science 241, 1210-1213. Pereira, L., Wolff, M.H., Fenwick, M. and Roizman, B. (19771 Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells. Virology 71, 733-749. Persson, R.H., Bacchetti, S. and Smiley, J.R. (1985) Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans. J. Virol. 54, 414-421. Poffenberger. K.L., Raichlen, P.E. and Herman, R.C. In vitro characterization of a herpes simplex virus type 1 ICP22 null mutant. Virus Genes, in press. Preston, CM., Frame, M.C. and Campbell, M.E.M. (1988) A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence. Cell 52, 425-434. Russell, .I., Stow, N.D., Stow, EC. and Preston, CM. (1987) Herpes simplex virus genes involved in latency in c&u. J. Gen. Viral. 68, 3~9-3~18. Spaete, R.R. and Mocarski, E.S. (1985) Regulation of cytomegalovirus gene expression: alpha and beta promoters are transactivated by viral functions in permissive human fibroblasts. J. Viral. 56, 135-143. Spector, D., Purves, F. and Roizman, B. (1990) Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome. Proc. Nat]. Acad. Sci. USA 87. 5268-5272. Spector, D., Purves, F. and Roizman, B. (1991) Roie of alpha-transducing factor (VP16) in the induction of alpha genes within the context of viral genomes. J. Viral. 65, 3504-3513. Stern, S., Tanaka, M. and Herr, W. (1989) The act-1 homeodomain directs formation of a multiproteinDNA complex with HSV transactivator VP16. Nature 341, 624-630. Sturm, R., Das, G. and Herr, W. (1988) The ubiquitous octamer binding act-1 contains a POU domain with a homeobox subdomain. Genes Dev. 2, 1582-1599. Triezenberg, S.J., Kingsbury, R.C. and M&night, S.L. (1988) Functional dissection of VP16 the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718-729. Turk, S.R., Kik, N.A., Birch, G.M., Cbiego Jr., D.J. and Shipman Jr., C. (1989) Herpes simplex virus type-l ribonucleotide reductase null mutants induce lesions in guinea pigs. Virology 173, 733-735. Wilcox, K.W., Kohn, A., Sklyanskaya, E. and Roizman, B. (1980) Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA.J. Virol. 33, 167-182. Watson, R.J., Preston, CM., and Clements, J.B. (1979) Separation and characterization of herpes simplex virus type 1 immediate early mRNAs. J. Viroi. 31, 42-52. Wymer, J.P., Chung, T.D., Chang, Y.N., Hayward, GS. and Aurelian, L. (1989) Identification of immediate-early-type cis-response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2. J. Virol. 63, 2773-2784. Xiao, P. and Capone, J.P. (1990) A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with act-1. Mol. Cetl. Biol. 10, 4974-4977. Yao, F. and Courtney, R.J. (1989) A major transcriptional regulatory protein (ICP41 of herpes simplex virus type 1 is associated with purified virions. J. Viral. 63, 3338-3344. Yao, F. and Courtney, R.J. (1992) Association of ICPO but not ICP27 with purified virions of herpes simplex virus type 1. Zhang, Y.F. and Wagner, E.K. (1987) The kinetics of expression of individual herpes simplex virus type 1 transcripts. Virus Genes 1, 49-60.
