In vitro processing of herpes simplex virus type 1 DNA replication intermediates by the viral alkaline nuclease, UL12

Herpes simplex virus type 1 (HSV-1) DNA replication intermediates exist in a complex nonlinear structure that does not migrate into a pulsed-field gel. Genetic evidence suggests that the product of the UL12 gene, termed alkaline nuclease, plays a role in processing replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, J. Virol. 70:2075-2085, 1996). In this study we have tested the hypothesis that alkaline nuclease acts as a structure-specific resolvase. Cruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of being resolved into linear duplexes as would be expected of a resolvase activity, the artificial cruciforms were degraded. DNA replication intermediates were isolated from the well of a pulsed-field gel ("well DNA") and treated with purified HSV-1 alkaline nuclease. Although alkaline nuclease can degrade virion DNA to completion, digestion of well DNA results in a smaller-than-unit-length product that migrates as a heterogeneous smear; this product is resistant to further digestion by alkaline nuclease. The smaller-than-unit-length products are representative of the entire HSV genome, indicating that alkaline nuclease is not inhibited at specific sequences. To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate that replicating DNA apparently contains no accessible double-stranded ends but does contain nicks and gaps. Our data suggest that UL12 functions at nicks and gaps in replicating DNA to correctly repair or process the replicating genome into a form suitable for encapsidation.     

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.     

Cellular protein interactions with herpes simplex virus type 1 oriS

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Induced extrusion of DNA from the capsid of herpes simplex virus type 1

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.     

The UL8 subunit of the herpes simplex virus type-1 DNA helicase-primase optimizes utilization of DNA templates covered by the homologous single-strand DNA-binding protein ICP8

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.     

Herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) enhances the ability of the viral DNA helicase-primase to unwind cisplatin-modified DNA

The herpes simplex virus type-1 UL5, UL8, and UL52 genes encode an essential heterotrimeric DNA helicase-primase that is responsible for concomitant DNA unwinding and primer synthesis at the viral DNA replication fork. The viral single-strand DNA-binding protein (ICP8) can stimulate DNA unwinding by the helicase-primase as a result of a physical interaction that is mediated by the UL8 subunit. In this study, we investigated the ability of the helicase-primase to unwind a fork-like substrate that contains an intrastrand d(GpG) DNA cross-link produced by the antitumor drug cisplatin. We also examined the ability of ICP8 to modulate the effect of the cisplatin lesion. The data show that the lesion inhibited the helicase-primase when located on the DNA strand along which it translocates. However, the lesion did not represent a permanent obstacle to its progression. In contrast, the adduct did not affect the helicase-primase when located on the opposite DNA strand. ICP8 specifically stimulated DNA unwinding by the helicase-primase. Coating concentrations of ICP8 were necessary for optimal unwinding of damaged DNA. Addition of competitor DNA to helicase reactions led to substantial reduction of DNA unwinding by the helicase-primase, suggesting that the enzyme is distributive. ICP8 did not abolish the competition, indicating that it did not stimulate the helicase by increasing its processivity. Rather, ICP8 may stimulate DNA unwinding and enable bypass of cisplatin damaged DNA by recruiting the helicase-primase to the DNA.     

Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.     

Loss of DNA loop supercoiling and organization in cells infected by herpes simplex virus type 1

In cells infected by herpesviruses, a sequence of nuclear changes during interphase, as well as chromosomal aberrations during mitosis, are commonly observed. These changes suggest the progressive modification of host-cell chromatin. Previous studies have shown that the early chromatin modifications in cells infected by herpes simplex virus type 1 (HSV1) are not due to extensive breakdown of host-cell DNA or disruption of the nucleosomal structure. We have previously shown that infection by HSV1 induces single-stranded breaks in the host-cell DNA early in the course of infection, and that such breaks lead to modifications in the higher-order structure of host-cell chromatin. Here we report that virus-induced DNA breaks produce permanent long-term effects on the state of supercoiling and organization of the nuclear DNA loops, comparable to the DNA loop disorganization produced by high (and irreparable) doses of ultraviolet radiation.     

Atypical herpes simplex virus encephalitis diagnosed by PCR amplification of viral DNA from CSF

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.     

Dimerization and activation of the herpes simplex virus type 1 protease

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.     

Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK-gC-) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO2) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK-gCEPO2), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK-gCEPO2 recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK-gCEPO2 virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK-gCEPO2 showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.     

Stromal keratitis induced by a unique clinical isolate of herpes simplex virus type 1

The antibody responses of 65 volunteers receiving an i.d. regimen (0.1 ml given at two sites on days 0, 3, 7 and 0.1 ml given at one site on days 30 and 90) were compared with the control group of 35 volunteers receiving the standard i.m. regimen. By day 14, seroconversion was observed in all vaccinees in both groups. Geometric Mean Titers remained higher than 0.5 IU/ml throughout the study period. At the end of the observation period on day 365, antibodies persisted in all subjects. The multisite i.d. PCEC regimen has been proved as immunogenic as the standard i.m. regimen. Both regimens were well tolerated. Thus, it would be the effective and cheapest available rabies post-exposure treatment using tissue culture vaccine.     

Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8+ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8+ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species-mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans-and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8+-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8+ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47- mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.     

Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.     

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.     

The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.