Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Herpes simplex virus 2 UL45 is a type II membrane protein

In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45.     

Pathogenicity of glycoprotein C-deficient herpes simplex virus 1 strain TN-1 which encodes truncated glycoprotein C

A clinical isolate of herpes simplex virus 1 (TN-1) from a stromal keratitis patient was found to be defective in the glycoprotein C (gC) gene (UL44), thus resulting in the production of truncated gC upon infection. To study the pathogenetic role of truncated gC, we prepared a recombinant LTN-8 derived from TN-1 with deletions of the 1.5 kilobase pairs of the gC gene including the initiation codon. A penetration assay revealed LTN-8 to be less efficient in its penetration ability than TN-1, the laboratory strain KOS and RTN-1-20-3, a recombinant derived from TN-1 with the KOS gC gene. The penetration of LTN-8 was facilitated by the addition of TN-1-infected culture medium. TN-1 virus preparations had no hemagglutinating activity. However, the animals infected with TN-1 did develop hemagglutination inhibition (HI) antibodies. The LTN-8-infected animals did not develop HI antibodies. The pathogenicity in BALB/c mice following either corneal, intraperitoneal or intracerebral inoculation did not significantly differ among TN-1, RTN-1-20-3 or LTN-8. Our results indicate that truncated gC was sufficient for the induction of HI antibodies and was also able to facilitate penetration in vitro. Although truncated gC might be a virulence factor acting as a decoy, both truncated gC and intact gC had little effect on the outcome following intracerebral, intraperitoneal or corneal inoculation.     

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.     

Herpes simplex virus 1 DNA cleavage/packaging: the UL28 gene encodes a minor component of B capsids

High injury rates in particular industries in Alberta, Canada were the impetus for government initiatives to control injury frequency and severity. Safety associations, a financial incentives program, research projects, and safety demonstrations substantially decreased injury rates within 5 years. Since then, companies manage their own performance, but undergo periodic audits.     

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.     

Structure-function analysis of the herpes simplex virus type 1 UL12 gene: correlation of deoxyribonuclease activity in vitro with replication function

Although the product of the UL12 gene of herpes simplex virus type 1 (HSV-1) has been shown to possess both exonuclease and endonuclease activities in vitro, and deletion of most of the gene within the viral genome results in inefficient production and maturation of infectious virions, the function of the deoxyribonuclease (DNase) activity per se in virus replication remains unclear. In order to correlate the in vitro and in vivo activities of the protein encoded by UL12, mutant proteins were tested for nuclease activity in vitro by a novel hypersensitivity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase 1 or micrococcal nuclease. All mutants which lacked DNase activity in vitro also failed to complement the replication of AN-1 in nonpermissive cells. Likewise, all mutants which contained HSV-1 DNase activity, as detected by the hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of particular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzyme in vitro, complemented the replication of AN-1 significantly less than the wild-type protein. The results suggest that DNase activity per se is required for efficient replication of HSV-1 in vivo. However, residues, including the N-terminal 126 amino acids, which are dispensable for enzymatic activity in vitro may facilitate the accessibility or activity of the protein in vivo.     

Physical and functional interactions between the herpes simplex virus UL15 and UL28 DNA cleavage and packaging proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.     

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.     

Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.     

Efficient gene transfer to human squamous cell carcinomas by the herpes simplex virus type 1 amplicon vector

We determined the diagnostic value of the EEG in young children with Angelman syndrome (AS) and Rett syndrome (RS). EEGs, recorded before 5 years of age, of 10 patients with AS, 10 with RS and 10 with mental retardation of other origin were studied blindly by two examiners for the presence of the following items: (A) 4-6 Hz rhythmic activity of over 200 microV; (B) 2-3 Hz frontal activity of 200-500 microV; (C) posterior spikes; (D) triphasic frontal waves; (E) central and/or centro-temporal spike-wave complexes; and (F) other epileptic discharges. Based on these items the EEGs were scored as AS (A-D); RS (E-F); or other. Examiners never made a mistake between AS and RS. One examiner labeled 6 of 10 AS cases correctly, the other 5; 4 (5) were characterized as 'other.' In RS cases 5 were labeled as 'other' by the first examiner and 3 by the second one. We conclude that EEG patterns of AS and RS are sufficiently different to help differentiate between AS and RS at a young age, which has a bearing on genetic counseling.     

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 equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.     

Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.