A conserved domain of herpes simplex virus ICP34.5 regulates protein phosphatase complex in mammalian cells

The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed alpha, beta, and gamma, whose synthesis is regulated in a cascade fashion. alpha products are synthesized first during infection, and they are required for synthesis of beta and gamma proteins. To examine the expression of several HSV-1 beta and gamma genes in the absence of alpha functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only beta and gamma genes (0.315 to 0.421 map units). We found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the beta and gamma classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 alpha gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 beta and gamma genes can be transcribed in the absence of alpha functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the alpha gene product ICP4.

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Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2977-2983.1986 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2977-2983 ◽

Cited By ~ 1

Author(s):

L Pilon ◽

Y Langelier ◽

A Royal

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Infection ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Mammalian Cells ◽

Hprt Gene ◽

Hprt Locus ◽

Simplex Virus

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.

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High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion

Molecular and Cellular Biology ◽

10.1128/mcb.1.8.743-752.1981 ◽

1981 ◽

Vol 1 (8) ◽

pp. 743-752

Author(s):

R M Sandri-Goldin ◽

A L Goldin ◽

M Levine ◽

J C Glorioso

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protoplast Fusion ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

High Frequency ◽

Mammalian Cells ◽

Frequency Transfer ◽

Simplex Virus

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

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Mechanism of copper-mediated inactivation of herpes simplex virus.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.4.812 ◽

1997 ◽

Vol 41 (4) ◽

pp. 812-817 ◽

Cited By ~ 34

Author(s):

J L Sagripanti ◽

L B Routson ◽

A C Bonifacino ◽

C D Lytle

Keyword(s):

Hydrogen Peroxide ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mammalian Cells ◽

Reducing Agents ◽

Reducing Agent ◽

Free Radical Scavengers ◽

Protective Effects ◽

Relative Level ◽

Simplex Virus

The inactivation of herpes simplex virus (HSV) by copper was enhanced by the following reducing agents at the indicated relative level: ascorbic acid > hydrogen peroxide > cysteine. Treatment of HSV-infected cells with combinations of Cu(II) and ascorbate completely inhibited virus plaque formation to below 0.006% of the infectious virus input, while it maintained 30% viability for the host mammalian cells. The logarithm of the surviving fraction of HSV mediated by 1 mg of Cu(II) per liter and 100 mg of reducing agent per liter followed a linear relationship with the reaction time, in which the kinetic rate constant for each reducing agent was -0.87 min(-1) (r = 0.93) for ascorbate, -0.10 min(-1) (r = 0.97) for hydrogen peroxide, and -0.04 min(-1) (r = 0.97) for cysteine. The protective effects of metal chelators and catalase, the lack of effect of superoxide dismutase, and the partial protection conferred by free-radical scavengers suggest that the mechanism of copper-mediated HSV inactivation is similar to that previously reported for copper-mediated DNA damage. The sensitivity exhibited by HSV to Cu(II) and reducing agents, particularly ascorbate, might be useful in the development of therapeutic antiviral agents.

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Oncogenic transformation of mammalian cells in vitro by proflavine-photoinactivated herpes simplex virus type 2

Cancer Letters ◽

10.1016/s0304-3835(75)98243-9 ◽

1975 ◽

Vol 1 ◽

pp. 319-325 ◽

Cited By ~ 6

Author(s):

Jui-Lien H. Li ◽

Fred Rapp

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Mammalian Cells ◽

Oncogenic Transformation ◽

Simplex Virus

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High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

Molecular and Cellular Biology ◽

10.1128/mcb.1.8.743 ◽

1981 ◽

Vol 1 (8) ◽

pp. 743-752 ◽

Cited By ~ 82

Author(s):

R M Sandri-Goldin ◽

A L Goldin ◽

M Levine ◽

J C Glorioso

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protoplast Fusion ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

High Frequency ◽

Mammalian Cells ◽

Frequency Transfer ◽

Simplex Virus

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

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Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells

Archives of Virology ◽

10.1007/s00705-008-0094-x ◽

2008 ◽

Vol 153 (6) ◽

pp. 1191-1195 ◽

Cited By ~ 6

Author(s):

Tomoaki Mori ◽

Yusuke Mineta ◽

Yasuhiro Aoyama ◽

Takashi Sera

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mammalian Cells ◽

Tegument Protein ◽

Simplex Virus ◽

Protein Vp22

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mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A

Journal of Virology ◽

10.1128/jvi.79.15.9651-9664.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9651-9664 ◽

Cited By ~ 85

Author(s):

Pinghui Feng ◽

David N. Everly ◽

G. Sullivan Read

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Translation Initiation ◽

Mammalian Cells ◽

Translation Initiation Factor ◽

Site Directed Mutagenesis ◽

Initiation Factor ◽

Virion Host Shutoff ◽

Host Shutoff ◽

Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.

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