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P A Schaffer

Publications and source records attributed to P A Schaffer.

At least 19 recordsLinked to original sources
PubMed2001

ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells.

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

Adenoviridae↗
PubMed2001

Origin binding protein-containing protein-DNA complex formation at herpes simplex virus type 1 oriS: role in oriS-dependent DNA replication.

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.

Animals↗
PubMed2001

Phosphorylation of the herpes simplex virus type 1 origin binding protein.

The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.

Animals↗
PubMed2001

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency.

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.

Acyclovir↗
PubMed2000

Persistent elevated expression of cytokine transcripts in ganglia latently infected with herpes simplex virus in the absence of ganglionic replication or reactivation.

Infection of mouse trigeminal ganglia by herpes simplex virus induces cytokine expression that persists long after infectious virus or viral antigens become undetectable. To examine mechanisms underlying this phenomenon, we used a thymidine kinase mutant, dlsptk, which fails to replicate in ganglia and does not reactivate upon ganglionic explant. Using quantitative reverse transcriptase-polymerase chain reaction assays, we found that levels of interferon-gamma and tumor necrosis factor-alpha transcripts in dlsptk-infected ganglia were lower than those in wild type-infected ganglia, but were significantly (eight- to 10-fold) higher than those in mock-infected ganglia from Day 3 to Day 100 postinfection. We also studied latency-associated transcript (LAT) negative mutants that exhibit increased expression of productive cycle transcripts in ganglia. Ganglia infected with these mutants contained levels of cytokine transcripts similar to those in wild type-infected ganglia; any increases in viral antigen expression mediated by the LAT deletion were not accompanied by increased cytokine expression. Thus, neither viral replication, the ability to reactivate, nor LAT expression in ganglia is required for persistent elevated cytokine expression. The results provide indirect evidence that low-level expression of viral productive cycle genes in neurons can provide signals that elicit cytokine expression.

Animals↗
PubMed2000

Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation.

The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, "aptamers," have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation.

Cell Line↗
PubMed2000

Optimized viral dose and transient immunosuppression enable herpes simplex virus ICP0-null mutants To establish wild-type levels of latency in vivo.

The reduced efficiency with which herpes simplex virus type 1 (HSV-1) mutants establish latent infections in vivo has been a fundamental obstacle in efforts to determine the roles of individual viral genes in HSV-1 reactivation. For example, in the absence of the "nonessential" viral immediate-early protein, ICP0, HSV-1 is severely impaired in its ability to (i) replicate at the site of inoculation and (ii) establish latency in neurons of the peripheral nervous system. The mouse ocular model of HSV latency was used in the present study to determine if the conditions of infection can be manipulated such that replication-impaired, ICP0-null mutants establish wild-type levels of latency, as measured by viral genome loads in latently infected trigeminal ganglia (TG). To this end, the effects of inoculum size and transient immunosuppression on the levels of acute replication in mouse eyes and of viral DNA in latently infected TG were examined. Following inoculation of mice with 2 x 10(3), 2 x 10(4), 2 x 10(5), or 2 x 10(6) PFU/eye, wild-type virus replicated in mouse eyes and established latency in TG with similar efficiencies at all four doses. In contrast, increasing the inoculum size of the ICP0-null mutants n212 and 7134 from 2 x 10(5) to 2 x 10(6) PFU/eye significantly decreased the levels of infectious virus detected in the tear films of mice from days 4 to 9 postinfection. In an attempt to establish the biological basis for this finding, the effect of viral dose on the induction of the host proinflammatory response was examined. Quantitative reverse transcription-PCR demonstrated that increasing the inoculum of 7134 from 2 x 10(4) to 2 x 10(6) PFU/eye significantly increased the expression of proinflammatory (interleukin 6), cell adhesion (intercellular adhesion molecule 1), and phagocyte-associated (CD11b) genes in mouse eyes 24 h postinfection. Furthermore, transient immunosuppression of mice with cyclophosphamide, but not cyclosporin A, significantly enhanced both the levels of acute n212 and 7134 replication in the eye and the levels of mutant viral genomes present in latently infected TG in a dose-dependent manner. Thus, the results of this study demonstrate that acute replication in the eye and the number of ICP0-null mutant genomes in latently infected TG can be increased to wild-type levels for both n212 and 7134 by (i) optimization of inoculum size and (ii) transient immunosuppression with cyclophosphamide.

Animals↗
PubMed2000

Roscovitine, a specific inhibitor of cellular cyclin-dependent kinases, inhibits herpes simplex virus DNA synthesis in the presence of viral early proteins.

We have previously shown that two inhibitors specific for cellular cyclin-dependent kinases (cdks), Roscovitine (Rosco) and Olomoucine (Olo), block the replication of herpes simplex virus (HSV). Based on these results, we demonstrated that HSV replication requires cellular cdks that are sensitive to these drugs (L. M. Schang, J. Phillips, and P. A. Schaffer. J. Virol. 72:5626-5637, 1998). We further established that at least two distinct steps in the viral replication cycle require cdks: transcription of immediate-early (IE) genes and transcription of early (E) genes (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Since Rosco inhibits HSV replication efficiently even when added to infected cells at 6 h postinfection, we postulated that cdks may also be required for viral functions that occur after E gene expression. In the study presented herein, we tested this hypothesis directly by measuring the efficiency of viral replication, viral DNA synthesis, and expression of several viral genes during infections in which Rosco was added after E proteins had already been synthesized. Rosco inhibited HSV replication, and specifically viral DNA synthesis, when the drug was added at the time of release from a 12-h phosphonoacetic acid (PAA)-induced block in viral DNA synthesis. Inhibition of DNA synthesis was not a consequence of inhibition of expression of IE or E genes in that Rosco had no effect on steady-state levels of two E transcripts under the same conditions in which it inhibited viral DNA synthesis. Moreover, viral DNA synthesis was inhibited by Rosco even in the absence of protein synthesis. In a second series of experiments, the replication of four HSV mutants harboring temperature-sensitive mutations in genes essential for viral DNA replication was inhibited when Rosco was added at the time of shift-down from the nonpermissive to the permissive temperature. Viral DNA synthesis was inhibited by Rosco under these conditions, whereas expression of viral E genes was not affected. We conclude that cellular Rosco-sensitive cdks are required for replication of viral DNA in the presence of viral E proteins. This requirement may indicate that HSV DNA synthesis is functionally linked to transcription, which requires cdks, or that both viral transcription and DNA replication, independently, require viral or cellular factors activated by Rosco-sensitive cdks.

Animals↗
PubMed1999

Transactivation of herpes simplex virus type 1 immediate-early gene expression by virion-associated factors is blocked by an inhibitor of cyclin-dependent protein kinases.

Initiation of productive infection by human herpes simplex virus type 1 (HSV-1) requires cell cycle-dependent protein kinase (cdk) activity. Treatment of cells with inhibitors of cdks blocks HSV-1 replication and prevents accumulation of viral transcripts, including immediate-early (IE) transcripts (26). Inhibition of IE transcript accumulation suggests that virion proteins, such as VP16, require functional cdks to activate viral transcription. In this report, we show that a cdk inhibitor, Roscovitine, blocks VP16-dependent IE gene expression. In the presence of Roscovitine, the level of virion-induced activation of a transfected reporter gene (the gene encoding chloramphenicol acetyltransferase) linked to the promoter-regulatory region of the ICP0 gene was reduced 40-fold relative to that of untreated samples. Roscovitine had little effect on the interaction of VP16 with VP16-responsive DNA sequences as measured by electrophoretic mobility shift assays. These data indicate that VP16-dependent activation of IE gene expression requires functional cdks and that this requirement is independent of the ability of VP16 to bind to DNA.

Cyclin-Dependent Kinases↗
PubMed1999

Transcription of herpes simplex virus immediate-early and early genes is inhibited by roscovitine, an inhibitor specific for cellular cyclin-dependent kinases.

Although herpes simplex virus (HSV) replicates in noncycling as well as cycling cells, including terminally differentiated neurons, it has recently been shown that viral replication requires the activities of cellular cyclin-dependent kinases (cdks) (L. M. Schang, J. Phillips, and P. A. Schaffer, J. Virol. 72:5626-5637, 1998). Since we were unable to isolate HSV mutants resistant to two cdk inhibitors, Olomoucine and Roscovitine (Rosco), we hypothesized that cdks may be required for more than one viral function during HSV replication. In the experiments presented here, we tested this hypothesis by measuring the efficiency of (i) viral replication; (ii) expression of selected immediate-early (IE) (ICP0 and ICP4), early (E) (ICP8 and TK), and late (L) (gC) genes; and (iii) viral DNA synthesis in infected cultures to which Rosco was added after IE or IE and E proteins had already been synthesized. Rosco inhibited HSV replication, transcription of IE and E genes, and viral DNA synthesis when added at 1, 2, or 6 h postinfection or after release from a 6-h cycloheximide block. Transcription of a representative L gene, gC, was also inhibited by Rosco under all conditions examined. We conclude from these studies that cellular cdks are required for transcription of E as well as IE genes. In contrast, steady-state levels of at least one cellular housekeeping gene were not affected by Rosco. The requirement of viral IE and E transcription for cellular cdks may reflect either a requirement for specific cdk-activated cellular and/or viral transcription factors or a more global requirement for cdks in the transcriptional activation of the viral genome.

Animals↗
PubMed1999

ICP22 and the UL13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of RNA polymerase II.

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550-5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription.

Animals↗
PubMed1998

Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection.

Viral infection is sometimes associated with the initiation or exacerbation of autoimmune disease, although the underlying mechanisms remain unclear. One proposed mechanism is that viral determinants that mimic host antigens trigger self-reactive T cell clones to destroy host tissue. An epitope expressed by a coat protein of herpes simplex virus-type 1 (HSV-1) KOS strain has now been shown to be recognized by autoreactive T cells that target corneal antigens in a murine model of autoimmune herpes stromal keratitis. Mutant HSV-1 viruses that lacked this epitope did not induce autoimmune disease. Thus, expression of molecular mimics can influence the development of autoimmune disease after viral infection.

Adoptive Transfer↗
PubMed1998

Cellular transcription factors enhance herpes simplex virus type 1 oriS-dependent DNA replication.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains three binding sites for the viral origin binding protein (OBP) flanked by transcriptional regulatory elements of the immediate-early genes encoding ICP4 and ICP22/47. To assess the role of flanking sequences in oriS function, plasmids containing oriS and either wild-type or mutant flanking sequences were tested in transient DNA replication assays. Although the ICP4 and ICP22/47 regulatory regions were shown to enhance oriS function, most individual elements in these regions, including the VP16-responsive TAATGARAT elements, were found to be dispensable for oriS function. In contrast, two oriS core-adjacent regulatory (Oscar) elements, OscarL and OscarR, at the base of the oriS palindrome were shown to enhance oriS function significantly and additively. Specifically, mutational disruption of either element reduced oriS-dependent DNA replication by 60 to 70%, and disruption of both elements reduced replication by 90%. The properties of protein-DNA complexes formed in gel mobility shift assays using uninfected and HSV-1-infected Vero cell nuclear extracts demonstrated that both OscarL and OscarR are binding sites for cellular proteins. Whereas OscarR does not correspond to the consensus binding site of any known transcription factor, OscarL contains a consensus binding site for the transcription factor Sp1. Gel mobility shift and supershift experiments using antibodies directed against members of the Sp1 family of transcription factors demonstrated the presence of Sp1 and Sp3, but not Sp2 or Sp4, in the protein-DNA complexes formed at OscarL. The abilities of OscarL and OscarR to bind their respective cellular proteins correlated directly with the efficiency of oriS-dependent DNA replication. Cooperative interactions between the Oscar-binding factors and proteins binding to adjacent OBP binding sites were not observed. Notably, Oscar element mutations that impaired oriS-dependent DNA replication had no detectable effect on either basal or induced levels of transcription from the ICP4 and ICP22/47 promoters, as determined by RNase protection assays. The Oscar elements thus appear to provide binding sites for cellular proteins that facilitate oriS-dependent DNA replication but have no effect on transcription of oriS-flanking genes.

Animals↗
PubMed1998

A virus with a mutation in the ICP4-binding site in the L/ST promoter of herpes simplex virus type 1, but not a virus with a mutation in open reading frame P, exhibits cell-type-specific expression of gamma(1)34.5 transcripts and latency-associated transcripts.

The herpes simplex virus type 1 L/S junction-spanning transcripts (L/STs) are a family of multisized transcripts expressed at high levels in cells infected with mutant viruses that (i) do not express ICP4, (ii) specify forms of ICP4 unable to bind to the consensus ICP4 binding site, or (iii) contain mutations in the ICP4 binding site located at the transcriptional start site of the L/STs. By extension, the failure to detect the L/STs in wild-type virus-infected cells is due to the repressive effect of ICP4 bound to its cognate binding site upstream of the L/ST transcription initiation site. ORF-P, the first and largest open reading frame (ORF) encoded by the L/STs, overlaps >90% of the ORF encoding ORF-34.5, a putative neurovirulence factor, which is transcribed from the opposite DNA strand. Viruses with mutations in the overlapping region of ORF-P and ICP34.5 exhibit premature shutoff of infected-cell protein synthesis and are highly attenuated following intracranial inoculation of juvenile mice. To determine whether the premature protein shutoff and neuroattenuated phenotypes of ORF-P ORF-34.5 double mutants are a consequence of alterations in ORF-P, ORF-34.5, or both, viruses containing mutations only in ORF-P or only in the ICP4 binding site in the L/ST promoter were isolated and characterized. Mutant virus L/ST-n38 contains a single-base-pair transition mutation in ORF-P codon 38, resulting in translational termination of the ORF-P protein (OPP). This mutation does not alter the amino acid sequence of ICP34.5. Expression of a truncated form of OPP by mutant virus L/ST-n38 did not result in premature shutoff of infected-cell protein synthesis and produced no other observable phenotype relative to wild-type virus in in vitro tests. Moreover, the 50% lethal dose (LD50) of L/ST-n38 was comparable to that of wild-type virus following intracranial inoculation of 3-week-old mice, as were the latency and reactivation phenotypes of the virus. These properties of L/ST-n38 indicate that the attenuated phenotype of ORF-P ORF-34.5 double mutants is a consequence of mutations that affect the function of ICP34.5 and not the function of OPP. Mutant virus LST-4BS contains four single-base-pair substitutions in the ICP4 binding site in the L/ST promoter that abrogate the binding of ICP4 to this site, leading to high-level expression of the L/STs and OPP. LST-4BS induced premature shutoff of viral and cellular protein synthesis and was slightly growth restricted in cells of neural lineage (SK-N-SH human neuroblastoma cells) but was wild type for these two parameters in cells of nonneural lineage (immortalized primate Vero cells). Of particular interest was the observation that L/ST-4BS exhibited cell-type-specific expression of both the gamma(1)34.5 transcripts and the latency-associated transcripts (LATs). Thus, expression of these transcripts was barely detectable in cells of neural lineage (NB41A3 mouse neuroblastoma cells) but was wild type in Vero cells. In vivo, L/ST-4BS was reactivated from mouse trigeminal ganglia with reduced efficiency and delayed kinetics relative to wild-type virus. L/ST-4BS was completely attenuated for neurovirulence (LD50 > 10(6) PFU) relative to wild-type virus (LD50 < 900 PFU), although the four single-base-pair substitutions lie outside the coding region for the neurovirulence factor, ICP34.5. Collectively, the complex in vitro and in vivo phenotypes of L/ST-4BS can be attributed to (i) disruptions of the ICP4 binding site in the L/ST promoter and subsequent overexpression of the L/STs and OPP; (ii) alterations in ORF-O, which is also mutated in L/ST-4BS; or (iii) alterations in other cryptic genes or cis-acting elements.

Amino Acid Sequence↗
PubMed1998

Characterization of a nerve growth factor-inducible cellular activity that enhances herpes simplex virus type 1 gene expression and replication of an ICP0 null mutant in cells of neural lineage.

Herpes simplex virus type 1 (HSV-1) ICP0 is required for efficient viral gene expression during lytic infection, especially at low multiplicities. A series of cellular activities that can substitute for ICP0 has been identified, suggesting that when the activity of ICP0 is limiting, these activities can substitute for ICP0 to activate viral gene expression. The cellular activities may be especially important during reactivation of HSV from neuronal latency when viral gene expression is initiated in the absence of prior viral protein synthesis. Consistent with this hypothesis, we have identified an inducible activity in cells of neural lineage (PC12) that can complement the low-multiplicity growth phenotype of an ICP0 null mutant, n212. Pretreatment of PC12 cells with nerve growth factor (NGF) or fibroblast growth factor (FGF) prior to infection produced a 10- to 20-fold increase in the 24-h yield of n212 but only a 2- to 4-fold increase in the yield of wild-type virus relative to mock treatment. Slot blot analysis of nuclear DNA isolated from infected cells treated or mock treated with NGF indicated that NGF treatment does not significantly affect viral entry. The NGF-induced activity in PC12 cells was expressed transiently, with peak complementing activity observed when cells were treated with NGF 12 h prior to infection. Addition of NGF 3 h after infection had little effect on virus yield. The NGF-induced cellular activity was inhibited by pretreatment of PC12 cells with kinase inhibitors that have high specificity for kinases involved in NGF/FGF-dependent signal transduction. RNase protection assays demonstrated that the NGF-inducible PC12 cell activity, like that of ICP0, functions to increase the level of viral mRNA during low-multiplicity infection. These results suggest that activation of viral transcription by ICP0 and transcriptional activation of cellular genes by NGF and FGF utilize common signal transduction pathways in PC12 cells.

Animals↗
PubMed1998

Requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription.

Several observations indicate that late-G1/S-phase-specific cellular functions may be required for herpes simplex virus (HSV) replication: (i) certain mutant HSV strains are replication impaired during infection of cells in the G0/G1 but not in the G1/S phase of the cell cycle, (ii) several late-G1/S-phase-specific cellular proteins and functions are induced during infection, and (iii) the activity of a cellular protein essential for expression of viral immediate-early (IE) genes, HCF, is normally required during the late G1/S phase of the cell cycle. To test the hypothesis that late-G1/S-phase-specific cellular functions are necessary for HSV replication, HEL or Vero cells were infected in the presence of the cell cycle inhibitors roscovitine (Rosco) and olomoucine (Olo). Both drugs inhibit cyclin-dependent kinase 1 (cdk-1) and cdk-2 (required for cell cycle progression into the late G1/S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G1). We found that HSV replication was inhibited by Rosco and Olo but not by lovastatin (a cell cycle inhibitor that does not inhibit cdk activity), staurosporine (a broad-spectrum protein serine-threonine kinase inhibitor), PD98059 (an inhibitor specific for erk-1 and -2) or iso-Olo (a structural isomer of Olo that does not inhibit cdk activity). The concentrations of Rosco and Olo required to inhibit cell cycle progression and viral replication in both HEL and Vero cells were similar. Inhibition of viral replication was found not to be mediated by drug-induced cytotoxicity. Efforts to isolate Rosco- or Olo-resistant HSV mutants were unsuccessful, indicating that these drugs do not act by inhibiting a single viral target. Viral DNA replication and accumulation of IE and early viral RNAs were inhibited in the presence of cell cycle-inhibitory concentrations of Rosco or Olo. We therefore conclude that one or more cdks active from late G1 onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.

Amino Acid Sequence↗
PubMed1997

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.

Animals↗