The TATGARAT box of the HSV-1 ICP27 gene is essential for immediate early expression but not critical for efficient replication in vitro or in vivo.

We constructed a recombinant virus containing a promoter mutation altering the immediate-early expression of the HSV-1 ICP27 transcript, ICP27DeltaSma, which contains a deletion of the "TATGARAT" and surrounding sequences, but retains the rest of the ICP27 promoter. This mutant does not exhibit immediate-early expression of ICP27 using criteria of expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. While transcript abundance at 1h after infection at 0.1 PFU/cell in mouse embryo fibroblasts was significantly altered compared to infections with wt -rescues, by 4 h after infection these differences were diminished or absent. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue infections at the earliest times tested, but were equivalent by 8-12 h pi. Further, both single and multi-step virus replication was equivalent with both mutants and rescues. Thus, altering the immediate early kinetics of ICP27 leads to a sub-optimal quantitative lag-phase in gene expression but without consequence to replication fitness in vitro . Infections in vivo also revealed the ability of mutant and rescue virus to invade the CNS of mice following footpad injections was equivalent. The nature of the role of immediate-early ICP27 expression is discussed in light of these observations.

Immediate-early expression of the herpes simplex virus type 1 ICP27 transcript is not critical for efficient replication in vitro or in vivo.

We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

Global analysis of herpes simplex virus type 1 transcription using an oligonucleotide-based DNA microarray.

More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early alpha27 (U(L)54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the alpha27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.

Self-interaction of the herpes simplex virus type 1 regulatory protein ICP27.

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a nuclear phosphoprotein required for viral lytic infection, which acts partly at the posttranscriptional level to affect RNA processing and export. In the present study, we show that ICP27 can interact with itself in vivo. Immunofluorescent staining of cells expressing both an ICP27 mutant with a deletion of the major nuclear localization signal and wild-type ICP27 showed that the mutant protein was efficiently imported into the nucleus in the majority of the cotransfected cells, suggesting heterodimer formation between the wild-type and mutant proteins. Coimmunoprecipitation experiments using epitope-tagged wild-type ICP27 and a series of ICP27 mutants with deletions and insertions in important functional regions of the protein revealed that the C-terminal cysteine-histidine-rich zinc-finger-like region of ICP27 was required for the self-association. Furthermore the self-association was also shown in yeast using two-hybrid assays, and again, an intact C-terminal zinc-finger-like region was required for the interaction. This study provides biochemical evidence that ICP27 may function as a multimer in infected cells.

Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27.

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.

Interactions between a herpes simplex virus regulatory protein and cellular mRNA processing pathways.

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP27 performs essential functions during viral lytic infection. Studies with viral mutants have demonstrated that ICP27 affects the shutoff of host protein synthesis, HSV-1 DNA replication, and the expression of viral early and late genes. Mounting evidence has been presented to demonstrate that ICP27 functions predominantly at the posttranscriptional level by affecting mRNA processing. That is, ICP27 alters poly(A) site usage, impairs host cell splicing, and facilitates the export of viral intronless mRNAs. These diverse effects occur by the interaction of ICP27 with viral and host proteins and by binding RNA. To define the precise mechanisms by which ICP27 affects RNA processing pathways, it is necessary to identify all of the molecular interactions of ICP27 in vivo and to determine the functional significance of these interactions. In vivo approaches will be emphasized here. Protein-protein interactions have been analyzed by coimmunoprecipitation studies, followed by immunoblotting to confirm the identity of coprecipitating proteins. Indirect immunofluorescence staining has been performed on cells treated with RNA polymerase II inhibitors to determine the intracellular distribution of ICP27 related to its RNA export function. Finally, in vivo UV irradiation has been used to covalently cross-link ICP27 to mRNAs in direct contact. This was followed with procedures to isolate and analyze the protein-RNA complexes. These studies have revealed several splicing complex proteins with which ICP27 interacts and have identified a number of intronless RNA transcripts to which ICP27 binds in the nucleus and cytoplasm in its role in RNA transport.

ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif.

Infection of metazoan cells with some viruses alters the balance of cellular mRNA export to favor viral RNA export and to retain cellular transcripts in the nucleus. Here, evidence is presented to show that the herpes simplex virus 1 (HSV-1) essential regulatory protein ICP27, which inhibits host cell-splicing, resulting in the accumulation of unspliced transcripts in the nucleus, mediates RNA export of viral intronless mRNAs. ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich nuclear export signal, which alone was able to direct the export of the heterologous green fluorescent protein. In vivo UV irradiation studies demonstrated that ICP27 could be crosslinked to poly(A)+ RNA in the nucleus and the cytoplasm, supporting a role in export. Furthermore, the amount of hnRNP A1, which has been implicated in the export of cellular spliced mRNAs, that was bound to poly(A)+ RNA in HSV-1-infected cells was reduced compared with uninfected cells. In addition, it was demonstrated that ICP27 bound seven intronless HSV-1 transcripts in both the nucleus and the cytoplasm, and export of these transcripts was diminished substantially during infection with an ICP27 null mutant virus. In contrast, ICP27 did not bind to two HSV-1 mRNAs that undergo splicing. Finally, binding of ICP27 to RNA in vivo required an arginine-glycine region that resembles an RGG box. These results indicate that ICP27 is an important viral export factor that promotes the transport of HSV-1 intronless RNAs.

Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is required posttranscriptionally for the expression of HSV-1 late genes during a productive infection. ICP27 also inhibits host cell pre-mRNA splicing, effectively shutting off host cell protein synthesis. Here we describe intragenic suppressors of LG4, a virus with a conditional lethal mutation in the gene encoding ICP27. At the restrictive temperature, tsICP27 from LG4 fails to inhibit host cell pre-mRNA splicing and to activate the expression of HSV-1 late-gene products. Although the suppressors of LG4 restore virus growth, they still fail to inhibit host cell pre-mRNA splicing. Thus, the role of ICP27 in the synthesis of late proteins is independent of host shutoff. In HSV-1-infected cells, ICP27 shuttles between the nucleus and the cytoplasm. Shuttling of ICP27 occurs only at late times during infection. In transfected cells, ICP27 shuttling was dependent on coexpression of RNA from a late HSV-1 gene. While shuttling does not occur in cells infected with LG4 at 39.5 degrees C, the suppressors of LG4 restore shuttling. Temperature shift experiments correlate the defect in shuttling with the temperature-sensitive phenotype of LG4. These data provide a correlation between shuttling of ICP27 and the expression of HSV-1 late-gene products. We propose that ICP27 regulates late-gene protein synthesis by facilitating the export of late RNAs.

The herpes simplex virus type 1 regulatory protein ICP27 coimmunoprecipitates with anti-Sm antiserum, and the C terminus appears to be required for this interaction.

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP27 is required for the inhibition of host cell splicing during viral infection and for the reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs). To determine what effect ICP27 had on splicing proteins that might cause their redistribution, we looked at proteins that were immunoprecipitated with anti-Sm antisera. No significant changes were seen in the migration or amounts of several snRNP common and snRNP-specific proteins from infected cells labeled with [35S]methionine, suggesting that the synthesis of these proteins was not altered by viral infection. However, when cells were labeled with 32Pi, differences were seen in the phosphorylation of at least two proteins depending on whether ICP27 was expressed. One protein, which had an apparent molecular mass of about 85 kDa, was highly phosphorylated during wild-type HSV-1 infection but much less so during infection with an ICP27 null mutant. The other protein, which migrated at the position of the U1 70-kDa protein and was precipitated with U1-specific antiserum, was also more highly phosphorylated when ICP27 was expressed during infection. Furthermore, a phosphoprotein with an apparent molecular mass of 63 kDa was found to coimmunoprecipitate with anti-Sm antisera during wild-type HSV-1 infection. ICP27 has an apparent molecular mass of 63 kDa, and immunoblot analysis confirmed that ICP27 coimmunoprecipitated with snRNPs. Analysis of mutations throughout the ICP27 protein demonstrated that the region that was required for this interaction was the C terminus of the protein, which includes a cysteine-histidine-rich region that resembles a zinc-finger-like motif. These data suggest that ICP27 interacts with snRNPs during infection and that it fosters changes in the phosphorylation state of at least two proteins that immunoprecipitate with snRNPs, although these studies do not demonstrate whether it does so directly or indirectly.

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.

Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect.

While the majority of metazoan genes and those of the DNA viruses which infect them contain introns which require RNA splicing, herpes simplex virus type 1 (HSV-1) encodes relatively few spliced products. We previously showed that the HSV-1 immediate-early protein ICP27 decreased the levels of spliced target mRNAs in transfections and spliced cellular mRNAs during infection, suggesting that ICP27 may function in impairing host cell splicing. Here, we show that during infections with the wild type, but not in infections with an ICP27 viral mutant termed 27-LacZ, precursor RNA accumulated for a virus transcript which contained introns. Pre-mRNA accumulation in the nucleus was greater than that in the cytoplasm, indicating that splicing rather than transport was affected. Furthermore, splicing of a beta-globin pre-mRNA substrate was inhibited in nuclear extracts from wild-type-infected cells but not in extracts from cells infected with 27-LacZ. The inhibitory activity in extracts from wild-type-infected cells was able to reduce the splicing efficiency of competent extracts in biochemical complementation assays. ICP27 appeared to be responsible for this decrease, because the splicing activity of an extract from cells infected with an ICP27 ts mutant was significantly reduced after incubation of the extract at the permissive temperature to allow renaturation of the conformationally defective ICP27 protein. These results strongly suggest that HSV-1 infection inhibits host cell splicing through the action of ICP27.

The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection.

We have previously shown that the herpes simplex virus immediate-early regulatory protein ICP27 acts posttranscriptionally to affect mRNA processing (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). Specifically, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. Here, we have investigated the effect of ICP27 during herpes simplex virus type 1 (HSV-1) infection on accumulation of spliced cellular mRNAs. ICP27 viral mutants have been shown to be defective in host shutoff (W. R. Sacks, C. C. Greene, D. P. Aschman, and P. A. Schaffer, J. Virol. 55:796-805, 1985). Therefore, we examined whether ICP27 could contribute to this complex process by decreasing cellular mRNA levels through its effects on host cell splicing. It was found that in infections with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs were higher than those seen with wild-type HSV-1. The differences occurred posttranscriptionally as shown by nuclear runoff transcription assays. The stabilities of the spliced products during infection with wild-type or ICP27 mutant viruses were similar, and unspliced precursor mRNA for a viral spliced gene was detected in infections with wild-type HSV-1 but not in infections in which ICP27 was not expressed. These results suggest that the reduction in cellular mRNA levels and the accumulation of pre-mRNA are related and may be caused by an impairment in host cell splicing. These data further show that ICP27 is required for these effects to occur.

Properties of an HSV-1 regulatory protein that appears to impair host cell splicing.

The herpes simplex virus type 1 (HSV-1) immediate early (alpha) protein ICP27 is an essential regulatory protein that appears to be involved in a number of different processes during lytic viral infection. Viral mutants defective in ICP27 have a variety of phenotypes that include defects in the shutoff of host protein synthesis, overexpression of some immediate early and early genes, reduced levels of DNA replication, and severely reduced levels of late gene products. ICP27 has been shown to act posttranscriptionally in the performance of some of its regulatory roles. It affects mRNA processing at the level of both polyadenylation and splicing. During polyadenylation, ICP27 appears to stimulate 3' RNA processing at selected poly(A) sites. The opposite effect, occurs on host cell splicing, that is, during HSV-1 infection, an inhibition in host cell splicing was found that required ICP27 expression. This impairment of splicing contributes to the shutoff of host protein synthesis by decreasing levels of spliced cellular mRNAs available for translation. A redistribution of splicing factors regulated by ICP27 has also been seen. Mutational analysis has shown that the C-terminal repressor region of the protein is required for the effects seen on splicing, whereas the activator region, encompassing the C-terminal half of the protein is required for the effects on 3' processing and the induction of late gene expression during viral infection. A highly basic arginine-rich region in the N-terminal half of the protein is required for nuclear localization of ICP27. Details on the mechanisms by which ICP27 contributes to these regulatory processes have been poorly defined to date.

Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing.

We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992).

The herpes simplex virus immediate early protein ICP27 encodes a potential metal binding domain and binds zinc in vitro.

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory proteins ICP27 and ICP0 each encode putative zinc-finger metal-binding domains. We utilized the technique of metal chelate affinity chromatography to demonstrate that ICP27 and ICP0 were able to bind to zinc in vitro. This property was further exploited to purify ICP27 from extracts of HSV-1-infected cells. The purification procedure also revealed that ICP27 possessed single-stranded DNA-binding activity. Analysis of ICP27 truncated peptides produced by in vitro translation verified that the zinc-binding region of ICP27 resides in the carboxy terminal 105 amino acids spanning the putative metal binding motif. However, a specific configuration of cysteine and histidine residues in this region was not required for binding to occur as demonstrated by the ability of a frame-shift mutation to bind with an efficiency similar to wild type. The mutated peptide retained four histidine and cysteine residues but in a configuration different from the consensus proposed for zinc-finger motifs. Therefore, while the region spanning the metal binding domain of ICP27 is essential for both the activator and repressor functions, and ICP27 binds zinc in vitro, it is not clear whether zinc binding in vivo is necessary for function.

A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing.

Post-transcriptional control mechanisms play an important role in regulating gene expression for a number of viruses, especially in the regulation of late gene products. In this study we have investigated the mode of action of ICP27, an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) required for late gene expression. Transfection experiments have demonstrated that ICP27 can activate or repress expression depending on the target gene. Here, we show that the regulatory activity of ICP27 is independent of the target gene promoter sequences but, instead, depends on the presence of different mRNA processing signals. The activation function correlated with different polyadenylation sites, whereas the repressor function correlated with the presence of introns either 5' or 3' to the target gene-coding sequences. Poly(A)+ RNA levels were increased by ICP27 in transfections with a target gene having only an AATAAA recognition signal but no G/U box within the usual distance. In contrast, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. These results suggest that this essential HSV-1 regulatory protein acts post-transcriptionally to affect mRNA processing and point to possible interactions between splicing and polyadenylation factors.

Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27.

The herpes simplex virus (HSV) immediate-early protein ICP27 has been postulated to play an auxillary role in HSV. gene expression, augmenting or inhibiting the activation of different viral promoters by the other immediate-early proteins ICPO and ICP4. Here we show that ICP27 alone can up-regulate gene expression of a retroviral vector containing Moloney murine leukemia virus (MoMuLV) regulatory sequences. This is the first report of an effect of ICP27 on gene expression driven by heterologous virus regulatory sequences. The effect does not involve the region of ICP27 which inhibits gene activation of HSV early gene promoters by ICPO and ICP4, but rather is dependent on the same region of ICP27 as is required to augment the activation of HSV late gene promoters by ICPO and ICP4. This indicates that the two activation effects are likely to operate via the same mechanism. Activation by ICP27 is dependent on the 3' LTR and adjacent region of MoMuLV but is independent of the promoter in the 5' LTR which can be replaced by a heterologous promoter such as that of SV40 without affecting activation by ICP27. The significance of this effect for an understanding of the mechanism of action of ICP27 and its role in regulating the gene expression of HSV and potentially of other viruses is discussed.

Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression.

The herpes simplex virus (HSV-1) immediate early protein ICP27 is a regulatory protein which is essential for virus replication. The phenotype of temperature-sensitive and deletion mutants in ICP27 includes overexpression of some immediate early and early gene products and greatly reduced levels of late gene products. To determine whether regulation by ICP27 occurs primarily at the transcriptional level, we have studied the expression of two immediate early products (ICP4 and ICP27) and two late gene products (glycoprotein B and glycoprotein C) at the level of transcription initiation, accumulation of steady state mRNA, and protein synthesis in an ICP27 temperature-sensitive mutant tsLG4, compared to wild-type HSV-1. At the nonpermissive temperature in tsLG4-infected cells, the two immediate early gene products ICP4 and ICP27 were overexpressed both at the mRNA and protein level although synthesis of these transcripts as measured by nuclear runoff assays was reduced relative to the wild-type HSV-1 infections. The transcription of late gene products glycoprotein B (gB) and glycoprotein C (gC) was lower in runoff assays from tsLG4 infections suggesting that the reduction in the level of late products occurred at the transcriptional level. However, temperature shift experiments in which tsLG4-infected cells were shifted to the nonpermissive temperature at various times after infection showed that the synthesis of late transcripts was not altered 2 hr after the shift whereas both the accumulation of leaky late and late mRNA and the incorporation of [35S]methionine into newly synthesized gB and gC was reduced by 2 hr after the shift to nonpermissive temperature. Therefore, while the synthesis of new transcripts continued, the accumulation of late mRNAs and their translation into protein was reduced when ICP27 was defective, whereas, the converse was found for immediate early products. That is, the synthesis of new transcripts was reduced yet mRNA and protein accumulated to high levels. These results suggest that ICP27 acts at least in part post-transcriptionally to regulate the expression of immediate early and late gene products.