Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

Mutational analysis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): characterization of HSV type 1 (HSV-1)/HSV-2 chimeras.

During lytic herpes simplex virus (HSV) infections, the half-lives of host and viral mRNAs are regulated by the HSV virion host shutoff (Vhs) protein (UL41). The sequences of the UL41 polypeptides of HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical. In spite of this similarity, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. To examine type-specific differences in Vhs function, we compared the Vhs activities of UL41 alleles from HSV-1(KOS) and HSV-2(333) by assaying the ability of a transfected UL41 allele to inhibit expression of a cotransfected reporter gene. Both HSV-1 and HSV-2 alleles inhibited reporter gene expression over a range of vhs DNA concentrations. However, 40-fold less of the HSV-2 allele was required to yield the same level of inhibition as HSV-1, indicating that it is significantly more potent. Examination of chimeric UL41 alleles containing various combinations of HSV-1 and HSV-2 sequences identified three regions of the 333 polypeptide which increase the activity of KOS when substituted for the corresponding amino acids of the KOS protein. These are separated by two regions which have no effect on KOS activity, even though they contain 43 of the 74 amino acid differences between the parental alleles. In addition, alleles encoding a full-length KOS polypeptide with a 32-amino-acid N-terminal extension retain considerable activity. The results begin to identify which amino acid differences are responsible for type-specific differences in Vhs activity.     

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.     

Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter

The parvovirus early protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene. We have examined the trans-activation of P38 by NS1 by using fusions of P38 to the reporter gene, chloramphenicol acetyltransferase (cat). Maximal trans-activation requires a small 5' cis element (tar) between -137 and -116. The tar element has activity in both orientations when 5' to the P38 promoter, but no activity has been detected 3' to the promoter. The wild-type P38 has a biphasic response to NS1 depending on the dosage of the NS1-expressing plasmid. Promoters lacking the tar also have a biphasic response that is reduced about 10-fold, and they can be inhibited by larger doses of the NS1 plasmid. Heterologous promoters from other viruses and the Harvey-ras oncogene promoter are inhibited by NS1. Truncated and internally deleted versions of NS1 lose the trans-activation, but some of them retain the inhibitory properties. Thus transactivation can be uncoupled from inhibition. The tar element has shown no activity with the heterologous simian virus 40 early promoter. In contrast, the P38 promoter responds to a heterologous enhancer, but the enhanced promoter loses activity to trans-activation by NS1. In summary, the P38 tar element has some of the properties of an enhancer with a high preference for a 5' position and a stringent requirement for the P38 promoter.     

The unique sequence of the herpes simplex virus 1 L component contains an additional translated open reading frame designated UL49.5.

We present evidence for the existence of an additional herpes simplex virus 1 gene designated UL49.5. The sequence, located between genes UL49 and UL50, predicts a hydrophobic protein with 91 amino acids. Attempts to delete UL49.5 were not successful. To demonstrate that UL49.5 is expressed, we made two recombinant viruses. First, we inserted in frame an oligonucleotide encoding a 15-amino-acid epitope known to react with a monoclonal antibody. This gene, consisting of the authentic promoter and chimeric coding domain, was inserted into the thymidine kinase gene of wild-type virus and in infected cells expressed a protein which reacted with the monoclonal antibody. The second recombinant virus contained a 5' UL49.5-thymidine kinase fusion gene. The protein expressed by this virus confirmed that the first methionine codon of UL49.5 served as the initiating codon. The predicted amino acid sequence of UL49.5 is consistent with the known properties of NC-7, a small capsid protein whose gene has not been previously mapped. A homolog of UL49.5 is present in the genome of varicella-zoster virus, located between homologs of UL49 and UL50.     

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.     

The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.     

An insertion vector for the analysis of gene expression during herpes simplex virus infection

A herpes simplex virus type 1 (HSV-1) insertion vector, pGal8, was designed for analysis of herpesvirus promoters during virus infection. This vector contains a multiple cloning site (MCS) positioned at the 5' end of the lacZ gene for the insertion of promoter sequences. The MCS and lacZ are flanked by sequences from the HSV-1 thymidine kinase encoding gene (tk) to direct homologous recombination into the tk locus of the viral genome. The utility of this vector is demonstrated by construction and comparison of recombinant viruses that express lacZ from the promoters of the genes encoding glycoprotein C, glycoprotein H and glycoprotein E.     

The herpes simplex virus UL37 protein is phosphorylated in infected cells.

The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation.     

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-1-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a gamma 1 gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCl gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication.     

The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.