UL9 Gene Research Articles

Role of human cytomegalovirus UL131A in cell type-specific virus entry and release

The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.

The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

Genetic Complexity of EHV-1 Defective Interfering Particles and Identification of Novel IR4/UL5 Hybrid Proteins Produced During Persistent Infection

This study examined the genetic complexity of three equine herpesvirus 1 (EHV-1) defective interfering particles (DIP) and found the DIP genomes to range from 5.9 kbp to 7.3 kbp in total size. Each DIP contains an identical 5' end ( approximately 1.9 kb) that harbors UL3 and UL4 genes that are 100% identical to those of the infectious virus. DIP2 and DIP3 contain a previously described unique IR4/UL5 (EICP22/EICP27) hybrid gene (Hyb1.0). The DIP1 genome, however, appears to be generated from a different recombination event which results in the formation of a new distinct hybrid ORF. The new ORF (Hyb2.0) is comprised of 684 bp from the 5' end of IR4 fused to 45 bp from the 3' terminus of UL5. In contrast to Hyb1.0, the UL5 sequences present in Hyb2.0 are not in-frame. Thus, the Hyb2.0 protein is comprised of 228 residues from IR4 linked to a sequence of 15 amino acids that result from a frameshifted reading of UL5 sequences. Western blot analysis confirmed that the Hyb2.0 ORF is expressed during persistent infection to produce a family of proteins that migrate at 36-42 kDa. Fluorescence microscopy revealed that both Hyb proteins display diffuse cytoplasmic localization patterns dissimilar to the nuclear localization patterns of both IR4 and UL5. Neither Hyb protein, however, disrupts the nuclear entry of the EHV-1 immediate-early, IR4, or UL5 proteins or cellular TATA box binding protein (TBP) previously shown to interact with both IR4 or UL5 in productive infection. DIP genomic segments ( approximately 3.5-5.0 kbp) downstream of the 100% conserved origin of replication are highly variable among the three DIP genomes and contain large areas of repetitive sequences. The possibility that the non-coding sequences play a role in viral interference and/or persistent infection remains to be determined.

Human cytomegalovirus (HCMV) UL82 gene product (pp71) relieves hDaxx-mediated repression of HCMV replication.

This study examines the role of the cellular protein hDaxx in controlling human cytomegalovirus (HCMV) immediate-early (IE) gene expression and viral replication. Using permissive cell lines that either overexpress hDaxx or are depleted of hDaxx expression by the use of short hairpin RNA, we demonstrate that hDaxx functions as a repressor of HCMV IE gene expression and replication. In addition, we demonstrate that the impaired growth phenotype associated with the UL82 (pp71) deletion mutant is abolished when hDaxx knockdown cells are infected, suggesting that pp71 functions to relieve hDaxx-mediated repression during HCMV infection.

The two helicases of herpes simplex virus type 1 (HSV-1)

Herpes simplex virus type 1 (HSV-1) encodes two helicases both of which are essential for viral DNA synthesis. UL9 binds specifically to the origins of replication and is believed to initiate DNA replication at one of three origins of replication located in the HSV-1 genome. The heterotrimeric helicase-primase complex, encoded by the UL5, UL8 and UL52 genes, is believed to unwind duplex viral DNA at replication forks and to prime lagging strand synthesis. Functional analyses of UL9 and the helicase-primase complex will be discussed with attention to the roles these proteins play during HSV-1 replication.

657. Combination Therapy with Cyclophosphamide and the Oncolytic Herpes Virus Mutant rRp450, Expressing a Cyclophosphamide Prodrug, Is Clinically Safe by Intratumoral, Intravenous, and Intracerebral Administration in an HSV-1 Susceptible Mouse Model

PURPOSE: Attenuated Herpes simplex virus (HSV) mutants are being actively pursued for cancer therapy. rRp450 is derived from the HSV-1 strain KOS, but is deleted for the UL39 gene (encoding ICP6, the large subunit of ribonucleotide reductase) and replaced by the rat CYP2B1 gene (encoding a cytochrome P450 enzyme that activates oxazophosphorines, e.g. cyclophosphamide). While cyclophosphamide has been shown to enhance antitumor efficacy of rRp450, safety studies of the combination have not been reported. We tested rRp450 for virus attenuation in primary cells and for toxicity in FVB/N mice following intratumoral, intravenous and intracerebral administration alone or in combination with systemic cyclophosphamide.

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

BackgroundOncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology.ResultsWe have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i) two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii) the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV) efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39 mutant).ConclusionOur Flip-Flop HSV-BAC system enables rapid generation of HSV vectors carrying transgene inserts. By introducing a tumor-specific-promoter-driven ICP4 cassette into pM24-BAC using this system, one should be able to generate transcriptionally-targeted oncolytic HSV vectors. We believe this system will greatly facilitate the screening of a plethora of clinically useful tumor-specific promoters in the context of oncolytic HSV vectors.

Human Cytomegalovirus Protein pp71 Disrupts Major Histocompatibility Complex Class I Cell Surface Expression

The human cytomegalovirus tegument protein pp71 is the product of the UL82 gene. Roles for pp71 in stimulating gene transcription, increasing infectivity of viral DNA, and the degradation of retinoblastoma family proteins have been described. Here we report a novel function for pp71 in limiting accumulation of cell surface major histocompatibility complex (MHC) class I complexes. MHC molecules were analyzed in glioblastoma cells exposed to a replication-defective adenovirus expressing UL82 (Adpp71) or after transient transfection of the UL82 gene. Accumulation of cell surface MHC class I levels diminished in a specific and dose-dependent manner after exposure to Adpp71 but not after exposure to an adenovirus expressing beta-galactosidase (Adbeta gal). UL82 expression did not interfere with accumulation of either MHC class I heavy-chain transcript or protein, nor did UL82 expression correlate with markers of apoptosis. Rather, UL82 expression correlated with an increased proportion of MHC class I molecules exhibiting sensitivity to endoglycosidase H treatment. Finally, we show that, in cells infected with recombinant virus strain missing all of the unique short region MHC class I evasion genes, disruption of UL82 expression by short, interfering RNAs led to increased accumulation of cell surface MHC class I complexes. These findings support a novel role for HCMV pp71 in disruption of the MHC class I antigen presentation pathway.

Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection

Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.

Inhibition of Anatid Herpes Virus-1 replication by small interfering RNAs in cell culture system

RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.

Characterization of an Antisense Transcript Spanning the UL81-82 Locus of Human Cytomegalovirus

In this study we present the characterization of a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR, UL81-82ast was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found. UL81-82ast was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.

Interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.

The human cytomegalovirus UL82-encoded pp71 protein is required for efficient virus replication and immediate-early gene expression when cells are infected at a low multiplicity. Functions attributed to pp71 include the ability to enhance the infectivity of viral DNA, bind to and target hypophosphorylated Rb family member proteins for degradation, drive quiescent cells into the cell cycle, and bind to the cellular protein hDaxx. Using UL82 mutant viruses, we demonstrate that the LXCXD motif within pp71 is not necessary for efficient virus replication in fibroblasts, suggesting that pp71's ability to degrade hypophosphorylated Rb family members and induce quiescent cells into the cell cycle is not responsible for the growth defect associated with a UL82 deletion mutant. However, UL82 mutants that cannot bind to hDaxx are unable to induce immediate-early gene expression and are severely attenuated for viral replication. These results indicate that the interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.

Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12

The alkaline nuclease (AN) encoded by gene UL12 of herpes simplex virus type 1 (HSV-1) is essential for efficient virus replication but its role during the lytic cycle remains incompletely understood. Inactivation of the UL12 gene results in reductions in viral DNA synthesis, DNA packaging, egress of DNA-containing capsids from the nucleus and ability of progeny virions to initiate new cycles of infection. Mechanistically, AN has been implicated in resolving branched structures in HSV-1 replicative intermediates prior to encapsidation, and promoting DNA strand-exchange. In this study, amplicons (bacterial plasmids containing functional copies of a virus replication origin and packaging signal) were used to analyse further the defects of the UL12 null mutant ambUL12. When ambUL12 was used as a helper virus both replication and packaging of the transfected amplicon were reduced in comparison with cells infected with wild-type (wt) HSV-1, and to extents similar to those previously observed for genomic ambUL12 DNA. By using amplicons differing at a specific restriction endonuclease site it was demonstrated that replicating molecules exhibit high frequency intermolecular recombination in both wt- and mutant-infected cells. Surprisingly, in the absence of the UL12 product, amplicons lacking a functional encapsidation signal were packaged. Moreover, these packaged molecules could be serially propagated indicating that they had been incorporated into functional virions. This difference in packaging specificity between wt HSV-1 and ambUL12 might indicate that replicative intermediates accumulating in the absence of AN contain an increased incidence of structures that can serve for the initiation of DNA packaging.

Identification of a monoclonal antibody from Peromyscus maniculatus (deer mouse) cytomegalovirus (PCMV) which binds to a protein with homology to the human CMV matrix protein HCMV pp71

In this study we identified and characterized a monoclonal antibody against the matrix protein of a cytomegalovirus isolated from the common deer mouse ( Peromyscus maniculatus) (PCMV). The monoclonal antibody was isolated using previously described technology which could be applied to the production of monoclonal antibodies against zoonotic disease. The antibody was found to react with a protein homologous to the human cytomegalovirus (HCMV) matrix protein (pp71), the product of the UL82 open reading frame (ORF). mAbs were generated from heterologous fusion of spleen cells from PCMV-positive mice and Balb/C P3X63-Ag8.653 myeloma cells. Using this approach, four monoclonal antibodies: B8C4, C12E8, G6A2 and P4E5 were generated. Antibody G6A2 reacted strongly with PCMV-infected cells as well as purified virions on ELISA and immunofluorescence. Western blot analysis, using sucrose gradient-purified virions, demonstrated that this mAb reacted specifically to a single protein with an apparent molecular weight of 71 kDa. The protein band was excised from the gel, purified and subjected to trypsin digestion followed by mass spectrometry. The protein sequences obtained were found to have identity to HCMV UL82 gene product. Sequence analysis indicated that it is the putative HCMV pp71 protein homolog of PCMV. G6A2 mAb did not cross-react with either human or murine recombinant pp71 proteins expressed in mammalian cells.

Employing tumor hypoxia to enhance oncolytic viral therapy in breast cancer

BackgroundHypoxia is a common tumor condition correlated with therapeutic resistance. Ribonucleotide reductase (RR) is a rate-limiting enzyme for viral replication. We hypothesize that hypoxia-driven transcription of UL39, the gene encoding the large subunit of RR, would enhance herpes oncolytic viral therapy in breast cancer. MethodsHypoxia-inducible factor 1α (HIF-1α) ELISA was performed on MCF7 human breast cancer cells in hypoxia (1% O2) or normoxia (21% O2). A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested in a luciferase assay. 10xHRE was cloned upstream of the UL39 gene (10xHRE-UL39). MCF7 cells were transfected with 10xHRE-UL39, incubated in hypoxia or normoxia, and infected with G207. Cytotoxicity assays and viral titers were performed. ResultsHIF-1α levels increased 7-fold in hypoxic MCF7 cells (P <.001). 10xHRE increased luciferase gene expression 61-fold in hypoxia versus controls (P <.01). G207 cytotoxicity of 10xHRE-UL39-transfected, hypoxic MCF7 cells increased 74% versus mock-transfected, hypoxic MCF7 cells (P <.001). In normoxia, 10xHRE-UL39 transfection did not significantly improve G207 cytotoxicity. 10xHRE-UL39 transfection improved peak viral titers 69-fold in hypoxia (P <.005), with no significant difference in normoxia. ConclusionHypoxia-driven RR production significantly enhances G207 cytotoxicity in hypoxic breast cancer cells, which would otherwise be resistant to herpes viral therapy alone.

Functional Properties of the Herpes Simplex Virus Type I Origin-binding Protein Are Controlled by Precise Interactions with the Activated Form of the Origin of DNA Replication

The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.

Nucleotide sequence of an ICP18.5 assembly protein (UL28) gene of green turtle herpesvirus pathogenically associated with green turtle fibropapilloma

Because newly identified green turtle herpesvirus (GTHV) is associated pathogenically with marine turtle fibropapillomatosis (FP) and it has not been isolated in vitro, molecular sequencing and analysis of the genomic DNA of this putative reptilian herpesvirus will enhance the current understanding of GTHV in causing the FP disease. An inverse polymerase chain reaction (IPCR) genomic walking technique was developed to obtain new DNA sequences based on a portion of known genomic sequence. Through two genomic walks, a 2169 bp DNA fragment of GTHV was cloned and sequenced. Sequence analysis shows that this DNA fragment contains the entire gene of the UL28, as well as the partial genomic sequence of the UL27 gene. The UL28 gene is 2250 bp long and encodes a 750-amino acid peptide known as ICP18.5 assembly protein of herpesviruses. Phylogenetic analysis of the GHTV UL28 gene showed a high sequence homology with the UL28 homologs of other herpesviruses and supports the current classification of GTHV to be a member of Alphaherpesvirinae. Identification of the genomic sequences of GTHV provides a molecular base for the development of diagnostic immunoassay and also for the determination of the pathogenic role of GTHV infection.

Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138 r3, K22 r5, and K22 r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22 r1 or K22 r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.

Implication of the product of the bovine herpesvirus type 1 UL25 gene in capsid assembly.

The UL25 ORF of bovine herpesvirus type 1 (BHV-1) was demonstrated recently to represent a gene encoding a 63 kDa viral protein. To investigate the role of this gene in virus replication, a BHV-1 UL25 deletion mutant was constructed. Although the UL25 mutant synthesizes late viral proteins and viral DNA, it fails to produce virus progeny in cells that do not express the UL25 gene, demonstrating that the UL25 protein is essential for the replicative cycle of BHV-1. Moreover, Southern blotting analyses of HindIII-digested DNA from infected non-complementing cells probed with the leftward terminal fragment of the BHV-1 linear genome revealed that the cleavage of the viral DNA produced is not impaired. However, the packaging of this cleaved DNA is compromised severely, since only few full C capsids were observed in infected non-complementing cells by transmission electron microscopy.