Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences.

Using an assay for recombination that measures deletion of a beta-galactosidase gene positioned between two directly repeated 350-bp sequences in plasmids transiently maintained in COS cells, we have found that replication from a simian virus 40 origin produces a high frequency of nonhomologous recombination. In contrast, plasmids replicating from a herpesvirus origin (oris) in COS cells superinfected with herpes simplex virus type 1 (HSV-1) show high levels of homologous recombination between the repeats and an enhanced recombinogenicity of the HSV-1 a sequence that is not seen during simian virus 40 replication. When the same assay was used to study recombination between 120- to 150-bp repeats in uninfected Vero cells, the level of recombination was extremely low or undetectable (< 0.03%), consistent with the fact that these repeats are smaller than the minimal efficient processing sequence for homologous recombination in mammalian cells. Recombination between these short repeats was easily measurable (0.5 to 0.8%) following HSV-1 infection, suggesting that there is an alteration of the recombination machinery. The frequency of recombination between repeats of the Uc-DR1 region, previously identified as the only segment of the HSV-1 a sequence indispensable for enhanced a-sequence recombination, was not significantly higher than that measured for other short sequences.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates.

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.     

Herpes simplex virus: selection of origins of DNA replication.

A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication. Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein. Plasmid libraries were generated using oligonucleotides with locally random sequences. The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1. Replicated plasmids resistant to Dpn I cleavage were amplified in E. coli. The selection scheme was repeated. Plasmids were isolated at different stages of the procedure and their replication efficiency was determined. Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region. In contrast, this was not seen using the box III library. We also noted that the wild type sequence invariably dominated the library after five rounds of selection. These plasmids arose from recombination between plasmids and viral DNA. Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication. In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator.     

Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.     

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.     

Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.

During the course of experiments designed to isolate deletion mutants of herpes simplex virus type 1 in the gene encoding the major DNA-binding protein, ICP8, a mutant, d61, that grew efficiently in ICP8-expressing Vero cells but not in normal Vero cells was isolated (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987). d61 was derived by cotransfection of ICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a spontaneous approximately 55-bp deletion in oriL. Gel electrophoresis and Southern blot analysis indicated that d61 DNA carried both deletions present in pDX. The ability of d61 to replicate despite the deletion in oriL suggested that a functional oriL is not essential for virus replication in vitro. Because d61 harbored two mutations, a second mutant, ts+7, with a deletion in oriL-associated sequences and an intact ICP8 gene was constructed. Both d61 and ts+7 replicated efficiently in their respective permissive host cells, although their yields were slightly lower than those of control viruses with intact oriL sequences. An in vitro test of origin function of isolated oriL sequences from wild-type virus and ts+7 showed that wild-type oriL, but not ts+7 oriL, was functional upon infection with helper virus. In an effort to determine the requirement for oriL in latency, ts+7 was compared with wild-type virus for its ability to establish, maintain, and be reactivated from latent infection in a murine eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after cocultivation with permissive cells, and each of the seven reactivated isolates was shown to carry the approximately 150-bp deletion characteristic of ts+7. These observations demonstrate that oriL is not required for virus replication in vitro or for the establishment and reactivation of latent infection in vivo.     

Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination.

The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination.     

Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment

The herpes simplex virus type 1 U(L)34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U(L)34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U(L)34 protein. This recombinant virus, in which the U(L)34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U(L)34 protein is essential for efficient envelopment of capsids.     

DNA mismatch repair proteins are required for efficient herpes simplex virus 1 replication

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of its human host cell and is known to interact with many cellular DNA repair proteins. In this study, we examined the role of cellular mismatch repair (MMR) proteins in the virus life cycle. Both MSH2 and MLH1 are required for efficient replication of HSV-1 in normal human cells and are localized to viral replication compartments. In addition, a previously reported interaction between MSH6 and ICP8 was confirmed by coimmunoprecipitation and extended to show that UL12 is also present in this complex. We also report for the first time that MLH1 associates with ND10 nuclear bodies and that like other ND10 proteins, MLH1 is recruited to the incoming genome. Knockdown of MLH1 inhibits immediate-early viral gene expression. MSH2, on the other hand, which is generally thought to play a role in mismatch repair at a step prior to that of MLH1, is not recruited to incoming genomes and appears to act at a later step in the viral life cycle. Silencing of MSH2 appears to inhibit early gene expression. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. The observation that MLH1 plays an earlier role in HSV-1 infection than does MSH2 is surprising and may indicate a novel function for MLH1 distinct from its known MSH2-dependent role in mismatch repair.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

Herpes simplex virus type 1 ICP8: helix-destabilizing properties.

The major single-stranded DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is one of seven virus-encoded polypeptides required for HSV-1 DNA replication. To investigate the role of ICP8 in viral DNA replication, we have examined the interaction of ICP8 with partial DNA duplexes and found that it can displace oligonucleotides annealed to single-stranded M13 DNA. In addition, ICP8 can melt small fragments of fully duplex DNA. Unlike a DNA helicase, ICP8-promoted strand displacement is ATP and Mg2+ independent and exhibits no directionality. It requires saturating amounts of ICP8 and is both efficient and highly cooperative. These properties make ICP8 suitable for a role in DNA replication in which ICP8 destabilizes duplex DNA during origin unwinding and replication fork movement.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate

Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.