Persistence of herpes simplex virus genes in cells of neuronal origin.

The growth characteristics of the KOS strain of herpes simplex virus type 1 (HSV-1) in cell lines of nervous tissues origin were examined in an attempt to develop a tissue culture system mimicking the in vivo state of HSV-1 latency. We have previously reported that the B103 rat brain neuroma cell line is nonpermissive for growth of the KOS strain. In this report, we show that this nonpermissiveness is a temperature- and multiplicity-dependent phenomenon, with minimum virus yields at an elevated temperature and a low multiplicity of infection. Under these conditions, B103 cells survived infection with active wild-type or mutant HSV-1, whereas similarly treated Vero cells were killed. Six independent cultures of B103 cells surviving HSV-1 infection have been established. The surviving cells ceased production of any HSV-1 virus by 14 days postinfection and resumed growth and division at rates comparable to those of uninfected B103 cells. Survivor cells continued to express HSV-1-specific antigens, however, as detected by indirect immunofluorescence and by surface iodination followed by immunoprecipitation and polyacrylamide gel electrophoresis. The survivor cells did not express all of the surface proteins seen on productively infected B103 cells, and they were not susceptible to complement-mediated immune cytolysis with anti-HSV-1 antiserum. These results demonstrate that at least a portion of the HSV-1 genome is being harbored in these survivor cells.     

Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) gene and a thymidine kinase deficient (tk-) mutant strain (HSV-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk- (143) and two tk+ (R970-5 and AC4) human cell lines. UV survival for each HSV-1 strain was similar for infection of both tk- and tk+ cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. These results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk.     

Genome sequence of herpes simplex virus 1 strain KOS

Herpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.     

Molecular genetic analysis of DNA polymerase and thymidine kinase genes of a HSV-1 population using an MPS technology

Real-time PCR was used to determine the ratio of viral and host DNA in lysates of Vero cells infected with HSV-1 strain L2. The number of virus copies reached a maximum after 24 h of incubation. Total isolated DNA was sequenced using the massively parallel sequencing technique on an Ion Torrent apparatus. Nucleotide sequences of thymidine kinase (UL23) and DNA polymerase (UL30) genes of a HSV-1 L2 population were determined; their primary structures were compared to those of other standard HSV-1 strains, KOS and 17. The detected differences between the UL23 and UL30 sequences of L2 and reference strains KOS and 17 were unimportant because these substitutions did not affect the conserved gene regions.     

Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Persistent infection with herpes simplex virus type 1 in an Ia antigen-positive murine macrophage cell line

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.     

Unstable heterozygosity in a diploid region of herpes simplex virus DNA

We have examined the behavior of a herpes simplex virus strain KOS isolate in which the two inverted repeats flanking the short segment of viral DNA differ in length by approximately 60 base pairs. We find that individual viral DNA molecules exist which contain the two distinguishable repeats, demonstrating that heterology between the repeats is tolerated. However, viruses heterozygous for the two different repeats are unstable, segregating both classes of homozygotes at a high frequency. We propose that this segregation is a consequence of the high-frequency recombination events which also result in genome segment inversion.     

A lambda library of Herpes simplex virus type 1 (KOS) DNA fragments obtained by partial digestion with Sau3A

Large fragments of Herpes simplex virus type 1 (HSV-1, strain KOS) DNA were produced by partial cleavage with Sau3A and inserted into a phage lambda BamHI vector. Recombinant phage (lambda KOS) DNA molecules were isolated and characterised. The final collection of phage recombinants contains partially overlapping inserts, which represent most of the HSV-1 genome. Restriction enzyme analysis of many independent clones containing Us sequences revealed sequence polymorphism in two specific regions.     

The herpes simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion

The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.     

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

The inverted repeats of IS<Emphasis Type="Italic">1384</Emphasis>, a newly described insertion sequence from<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> strain H,represent the specific target for integration of IS<Emphasis Type="Italic">1383</Emphasis>

Analysis of a region on plasmid pPGH1 from Pseudomonas putida strain H that is flanked by two copies of IS1383 has revealed an additional element with the typical features of a bacterial insertion sequence. This new IS element, designated IS1384, contains a single ORF of 972 bp, and is flanked by 9-bp inverted repeats. Based on sequence homology and structural characteristics of the putative transposase it encodes, IS1384 belongs to the IS5 subgroup of the IS5 family. Two copies of IS1384 are present on plasmid pPGH1, whereas none could be detected on the chromosome of P. putida strain H. Sequence analysis revealed the presence of two truncated copies of IS1384 on the second plasmid in this strain, pPGH2. The inverted repeats of all IS1384 copies (including the truncated ones) are interrupted by the integration of an IS1383 element. All integrations were found to be site- and orientation-specific. PCR studies and sequence data indicate that IS1383 can form a circular intermediate on excision. In the circular form, the previously described 13-bp inverted repeats of IS1383 are separated by 10 bp that are identical to the 5-bp motif that flanks each side of the element when it is integrated in its target. We provide evidence that these additional nucleotides, although not of inverted symmetry, represent an essential part of the inverted repeats. Furthermore, the data indicate that IS1383 integrated into the inverted repeats of IS1384 by a site-specific recombination rather than a site-specific insertion event.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness

Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.     

The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome.

We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.     

Evidence that the immediate-early gene product ICP4 is necessary for the genome of the herpes simplex virus type 1 ICP4 deletion mutant strain d120 to circularize in infected cells

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans.     

Activation and inactivation of Pseudomonas stutzeri methylbenzene catabolism pathways mediated by a transposable element

The arrangement of the genes involved in o-xylene, m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed ISPs1, which inactivates the m-xylene and p-xylene catabolic pathway in P. stutzeri OX1 and the o-xylene catabolic genes in P. stutzeri M1, was detected. No IS was detected in the corresponding catabolic regions of the P. stutzeri R1 genome. ISPs1 is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.