Nigel D. Stow

Isolation and Characterization of a Herpes Simplex Virus Type 1 Mutant Containing a Deletion within the Gene Encoding the Immediate Early Polypeptide Vmw110

Transfection experiments with plasmids containing immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) have previously demonstrated a role for the IE polypeptide Vmw110 (ICP0) in stimulating expression from plasmid-encoded early gene promoters. To gain further insights into the function of Vmw110 we isolated a deletion mutant specifying a truncated form of the polypeptide which had been shown to be inactive in transfection assays. This mutant, dl1403, contained a 2 kb deletion within both the TRL and IRL copies of the Vmw110 gene, and encoded a polypeptide consisting of the original N-terminal 105 amino acids followed by 56 amino acids specified by a reading frame not used by Vmw110. dl1403 was able to replicate and produce plaques on baby hamster kidney (BHK) cells but the yield of infectious virus was 20- to 100-fold lower than obtained with wild-type HSV-1. Surprisingly, comparison of polypeptide synthesis, DNA replication and DNA encapsidation in cells infected with 5 p.f.u./cell dl1403 or wild-type HSV-1 revealed no significant differences. In addition similar numbers of particles were produced in cells infected with the two viruses, resulting in stocks of dl1403 exhibiting significantly higher particle/p.f.u. ratios. The efficiency of plaquing of dl1403 was greatly reduced in Vero and human foetal lung cells compared with BHK cells, but following infection with 5 p.f.u./cell similar yields of infectious virus were obtained from all three cell lines. Marker rescue experiments verified that the reduced yield of dl1403 in BHK cells was a consequence of the deletion within the Vmw110 gene. The results suggest that the effect of this deletion is manifest primarily at low multiplicities of infection and can be largely overcome by increasing the virus dose.

Packaging of Genomic and Amplicon DNA by the Herpes Simplex Virus Type 1 UL25-Null Mutant KUL25NS

ABSTRACT The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060–1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into the cytoplasm.

The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein.

The products of herpes simplex virus type 1 (HSV-1) genes UL5, UL8 and UL52 form a complex in virus-infected cells that exhibits both DNA helicase and DNA primase activities. UL8 protein was purified from insect cells infected with a recombinant baculovirus and used to generate monoclonal antibodies (MAbs). MAb 0811 was shown to recognize the UL8 protein in both Western blots and immunoprecipitation assays and to co-precipitate the other two proteins in the complex from insect cells triply infected with recombinants expressing the UL5, UL8 and UL52 polypeptides. Experiments performed using extracts from doubly infected cells indicated that UL8 could interact separately with both the UL5 and UL52 proteins. Similar experiments using a recombinant virus that expressed the HSV-1 origin-binding protein (OBP), UL9, demonstrated a direct physical interaction between the helicase-primase complex and OBP which involved the UL8 subunit. The C-terminal DNA-binding domain of OBP is dispensable for this interaction, as evidenced by the ability of MAb 0811 to co-precipitate a truncated UL9 protein, containing only the N-terminal 535 amino acids, with UL8.

Identification of a varicella-zoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products

We have identified and characterized an origin of DNA replication in the genome of the human herpesvirus, varicella-zoster virus (VZV). This origin of replication (VZV ORIS) is located within the major inverted repeats in a position equivalent to that occupied by one of the herpes simplex virus type 1 (HSV-1) replication origins. Products encoded by both VZV and HSV-1 activate cloned copies of VZV ORIs, generating high molecular weight molecules consisting of tandem duplications of the input plasmid. The VZV ORIS region contains a tract of alternating A and T residues located at the centre of symmetry of an almost perfect palindrome of 45 bp, and the use of plasmid deletion mutants has demonstrated that this tract is an important functional element of the origin. Two sequences common to the VZV ORIS region and the regions specifying the two HSV-1 origins (ORIS, located within the TRS/IRS regions, and ORIL, located within the UL region) were identified and these may represent important recognition sites. One is an 11 bp sequence (CGTTCGCACTT), and the other is represented by the tract of alternating A and T residues. VZV does not appear to contain an origin of replication in a position equivalent to that of HSV-1 ORIL.

Herpes Simplex Virus Type 1 DNA-Packaging Protein UL17 Is Required for Efficient Binding of UL25 to Capsids

ABSTRACT Herpes simplex virus type 1 packages its DNA genome into a precursor capsid, referred to as the procapsid. Of the three capsid-associated DNA-packaging proteins, UL17, UL25, and UL6, only UL17 and UL6 appear to be components of the procapsid, with UL25 being added subsequently. To determine whether the association of UL17 or UL25 with capsids was dependent on the other two packaging proteins, B capsids, which lack viral DNA but retain the cleaved internal scaffold, were purified from nonpermissive cells infected with UL17, UL25, or UL6 null mutants and compared with wild-type (wt) B capsids. In the absence of UL17, the levels of UL25 in the mutant capsids were much lower than those in wt B capsids. These results suggest that UL17 is required for efficient incorporation of UL25 into B capsids. B capsids lacking UL25 contained about twofold-less UL17 than wt capsids, raising the possibilities that UL25 is important for stabilizing UL17 in capsids and that the two proteins interact in the capsid. The distribution of UL17 and UL25 on B capsids was examined using immunogold labeling. Both proteins appeared to bind to multiple sites on the capsid. The properties of the UL17 and UL25 proteins are consistent with the idea that the two proteins are important in stabilizing capsid-DNA structures rather than having a direct role in DNA packaging.

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear mini-chromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

Herpes simplex virus type 1 origin-dependent DNA replication in insect cells using recombinant baculoviruses.

The minimal set of seven herpes simplex virus type 1 (HSV-1) genes required for viral origin-dependent DNA synthesis was previously identified using a transient replication assay in a mammalian cell line permissive for HSV-1 growth. We have constructed recombinant baculoviruses which efficiently express the products of each of these seven genes in infected Spodoptera frugiperda (Sf) insect cells. When Sf cells were transfected with a plasmid containing a functional HSV-1 origin of replication, and subsequently superinfected with a mixture of these seven viruses, the input plasmid was amplified. This amplification exhibited properties characteristic of genuine HSV-1 DNA replication: all seven HSV-1 replication gene products were required, replicated DNA was detected as concatemers, and mutated origins were impaired to similar extents in insect cells and cells permissive for HSV-1 replication. These results demonstrate that the HSV-1 proteins expressed in Sf cells are fully competent for viral DNA synthesis, and indicate that any host function essential in mammalian cells must also be present in the infected insect cells. This system also provides a convenient method by which mutated replication proteins can be screened for function and produced in amounts sufficient for biochemical studies. Using this approach we show that the ability of the UL9 protein to bind to the viral origins of replication is not sufficient for it to facilitate DNA synthesis.

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28.

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.

New Genes from Old: Redeployment of dUTPase by Herpesviruses

ABSTRACT Published work (D. J. McGeoch, Nucleic Acids Res. 18:4105-4110, 1990; J. E. McGeehan, N. W. Depledge, and D. J. McGeoch, Curr. Protein Peptide Sci. 2:325-333, 2001) has indicated that evolution of dUTPase in the class of herpesviruses that infect mammals and birds involved capture of a host gene followed by a duplication event that resulted in a coding region comprising two fused dUTPase domains. Some of the conserved residues required for enzyme activity were then lost, resulting in a dUTPase containing a single active site with different elements contributed by each half of the protein. Further conserved residues were lost in one subfamily (the Betaherpesvirinae), yielding a protein that is related to herpesvirus dUTPases but has a different and as yet unrecognized function. Evidence from sequence similarities and structural predictions now indicates that several additional genes were derived from the herpesvirus dUTPase gene, probably by duplication. These are UL31, UL82, UL83, and UL84 in human cytomegalovirus (and counterparts in other members of the Betaherpesvirinae) and ORF10 and ORF11 in human herpesvirus 8 (and counterparts in other members of the Gammaherpesvirinae). The findings clarify the evolutionary history of these genes and provide novel insights for structural and functional studies.

Herpes Simplex Virus Genes Involved in Latency in vitro

The properties of temperature-sensitive (ts), insertion or deletion mutants of herpes simplex virus (HSV) were investigated in an in vitro model system for latency. The studies defined virus gene products required for establishment of latency and for reactivation of latent virus. All mutants tested established latency in human foetal lung fibroblasts and could be reactivated by intertypic superinfection with HSV or with human cytomegalovirus. Two mutants of HSV type 1 used in these studies, tsK and in1411, failed to synthesize active immediate early (IE) polypeptide Vmw175 and were blocked at a very early stage of the virus replication cycle, showing that, at most, only limited gene expression is necessary for the establishment of latency. Mutant dl1403, which lacks the gene encoding IE polypeptide Vmw110, established latency as efficiently as wild-type HSV. Latent HSV type 2 was reactivated by superinfection with tsK or in1411 but not with dl1403, suggesting that polypeptide Vmw110, which is known to regulate gene expression by trans-activation, is required for reactivation in the in vitro system.