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.

Download Full-text

Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2004 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2004-2010 ◽

Cited By ~ 28

Author(s):

A Graessmann ◽

G Sandberg ◽

E Guhl ◽

M Graessmann

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Synthesis ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Coding Region ◽

Simplex Virus

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

Download Full-text

Successful Retroviral Gene Transfer of Simian Virus 40 T Antigen and Herpes Simplex Virus-Thymidine Kinase into Human Hepatocytes

Cell Transplantation ◽

10.3727/000000001783986585 ◽

2001 ◽

Vol 10 (4-5) ◽

pp. 377-381 ◽

Cited By ~ 6

Author(s):

Naoya Kobayashi ◽

Hirofumi Noguchi ◽

Karen A. Westerman ◽

Takamasa Watanabe ◽

Toshihisa Matsumura ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Gene Transfer ◽

Thymidine Kinase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Human Hepatocytes ◽

Retroviral Gene Transfer ◽

Simplex Virus

Download Full-text

Gonadal tumors of mice double transgenic for inhibin-alpha promoter-driven simian virus 40 T-antigen and herpes simplex virus thymidine kinase are sensitive to ganciclovir treatment

Journal of Endocrinology ◽

10.1677/joe.0.1700079 ◽

2001 ◽

Vol 170 (1) ◽

pp. 79-90 ◽

Cited By ~ 8

Author(s):

MK Mikola ◽

NA Rahman ◽

TH Paukku ◽

PM Ahtiainen ◽

TE Vaskivuo ◽

...

Keyword(s):

Body Weight ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Somatic Cell ◽

Thymidine Kinase ◽

Fusion Gene ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Simplex Virus

We have previously produced transgenic (TG) mice expressing the mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (Inhalpha/Tag) fusion gene. The mice develop gonadal somatic cell tumors at the age of 5-7 months; the ovarian tumors originate from granulosa cells, and those of the testes from Leydig cells. In the present study another TG mouse line was produced, expressing under the same inh-alpha promoter the herpes simplex virus thymidine kinase gene (Inhalpha/TK). Crossbreeding of the two TG mouse lines resulted in double TG mice (Inhalpha/TK-Inhalpha/Tag), which also developed gonadal tumors. The single (Inhalpha/Tag) and double TG (Inhalpha/TK-Inhalpha/Tag) mice, both bearing gonadal tumors, were treated at the age of 5.5-6.5 months with ganciclovir (GCV, 150 mg/kg body weight twice daily i.p.) for 14 days, or with aciclovir (ACV, 300-400 mg/kg body weight per day perorally) for 2 months. During GCV treatment, the total gonadal volume including the tumor, decreased in double TG mice by an average of 40% (P<0.05), while in single TG mice, there was a concomitant increase of 60% in gonadal size (P<0.05). GCV was also found to increase apoptosis in gonads of the double TG mice. Peroral treatment with ACV was less effective, it did not reduce significantly the gonadal volume. We also analyzed the in vitro efficacy of ACV and GCV treatments in transiently HSV-TK-transfected KK-1 murine granulosa tumor cells, originating from a single-positive Inhalpha/Tag mouse. GCV proved to be more effective and more specific than ACV in action. These results prove the principle that targeted expression of the HSV-TK gene in gonadal somatic cell tumors is potentially useful for tumor ablation by antiherpes treatment. The findings provide a lead for further development of somatic gene therapy for gonadal tumors.

Download Full-text

Mutational Analysis of the Herpes Simplex Virus Triplex Protein VP19C

Journal of Virology ◽

10.1128/jvi.80.3.1537-1548.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1537-1548 ◽

Cited By ~ 24

Author(s):

Walt E. Adamson ◽

David McNab ◽

Valerie G. Preston ◽

Frazer J. Rixon

Keyword(s):

Amino Acids ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Insertional Mutagenesis ◽

Fluorescent Protein ◽

Mutational Analysis ◽

Simian Virus 40 ◽

T Antigen ◽

Virus Growth ◽

Simplex Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal ∼100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.

Download Full-text

A Novel Cellular Protein, p60, Interacting with both Herpes Simplex Virus 1 Regulatory Proteins ICP22 and ICP0 Is Modified in a Cell-Type-Specific Manner and Is Recruited to the Nucleus after Infection

Journal of Virology ◽

10.1128/jvi.73.5.3810-3817.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3810-3817 ◽

Cited By ~ 35

Author(s):

Renato Bruni ◽

Beatrice Fineschi ◽

William O. Ogle ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cellular Protein ◽

Wild Type Virus ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Rabbit Skin ◽

Skin Cells ◽

Genes Encoding ◽

Simplex Virus

ABSTRACT Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (γ2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent M r of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525–8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent M r but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent M r. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.

Download Full-text