Differential Substrate Specificity of DNA Polymerase beta and of a DNA Polymerase Induced by Herpes Simplex Virus Type 2 towards Thymidine Triphosphate Analogues

Kowalzick, L.; Gauri, K. K.; Spadari, Silvio; Pedrali-Noy, G.; Koch, G.

doi:10.1099/0022-1317-65-3-467

Cited by 5 publications

(3 citation statements)

References 18 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, the 13 genes have been assigned functions related to nucleotide metabolism and viral DNA synthesis. An imbalance, or excess, of these activities may lead to wastage and altered specificity (34)(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

“…The derivation of the vhs-i mutant was described previously (15). Virus stocks representing cell lysates were prepared by three cycles of freeze-thawing of infected cells (16), and virions were purified in dextran T 10 gradients (17,18 [24][25][26][27][28][29][30][31][32][33][34][35][36] hr, the blots were rinsed three times at room temperature with 2x NaCl/Cit, once (for 5-6 hr) at 70'C in 2x NaCl/Cit and 0.1% NaDodSO4, and three times in 0.2x NaCl/Cit at room temperature.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

Kwong¹,

Frenkel²

1987

Proc. Natl. Acad. Sci. U.S.A.

262

285

View full text Add to dashboard Cite

The herpes simplex virus viion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (a)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and a mRNAs, whereas purified vhs-I mutant virions lack this activity. The functional half-life of many early ((3)-and late (y)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscrininately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection.Cells infected with herpes simplex viruses 1 and 2 proceed through the sequential transcription of several coordinately regulated groups of viral genes, including the a (immediate early), A3 (delayed early), and yj and y2 (late) genes (1-3). As suggested originally by Honess and Roizman (1), this transcriptional regulation must also be accompanied by a negative translational-regulatory scheme. Thus, the translation of host mRNA is turned off soon after virus entry into the cells, and the synthesis of a-and (3-viral polypeptides decreases at later intervals after infection.The early shutoff of host-protein synthesis (4) (15). Surprisingly, the vhs mutants are also altered with respect to a function that is present in the wild-type (wt) virus inoculum, which decreases the synthesis of a proteins from preexisting a mRNA (15). These findings suggest that a single virion-associated function might limit the translation of both host and a mRNAs.In this paper, we present data concerning the specificity of the host shutofffunction. We show that purified virions of the wt virus, but not those of the vhs-i mutant virus, carry a function that reduces the half-life of host and a mRNAs.Furthermore, the functional half-life of many (-and y-viral mRNAs is also prolonged in vhs-i-infected cells. These findings suggest that wt virions contain a function that indiscriminately reduces the half-life of the majority of infected-cell mRNAs. EXPERIMENTAL PROCEDURESCells and Viruses. Mouse Ltk-cells were obtained from B. Roizman (University of Chicago) and Vero monkey cells were obtained from S. Bachenheimer (University of North Carolina). The derivation of the vhs-i mutant was described previously (15). Virus stocks representing cell lysates were prepared by three cycles of freeze-thawing of infected cells (16), and virions were purified in dextran T 10 gradients (17,18).Drug Treatment and Analyses of Proteins. For cycloheximide reversal in the presence of actinomycin D (1), the cells were incubated fo...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

Kwong¹,

Frenkel²

1987

Proc. Natl. Acad. Sci. U.S.A.

262

285

View full text Add to dashboard Cite

show abstract

“…Antiviral properties of EdU are probably mediated by the HSV-encoded TK. After selective phosphorylation of the drug by the viral TK and further phosphorylation by cellular enzymes, EdU-5'-triphosphate may incorporate into virus DNA as counterfeit thymidine, although the evidence for this is not conclusive (19,26). To confirm the role of the viral TK in activating this drug, EdU-resistant HSV variants were prepared in cell culture.…”

Section: Discussionmentioning

confidence: 99%

Antiviral activity of 5-ethyl-2'-deoxyuridine against herpes simplex viruses in cell culture, mice, and guinea pigs

Schinazi

Scott

Peters

et al. 1985

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The susceptibility of 3 laboratory strains and 24 clinical isolates of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to 5-ethyl-2'-deoxyuridine was determined in plaque reduction assays in Vero cells. The median effective doses were 8.6 and 7.8 ,M, respectively. The drug was less potent than acyclovir and other related antiviral drugs, but it had a high therapeutic index against both HSV-1 and HSV-2. Drug-resistant viruses were readily produced in cell culture. These variants were cross-resistant to acyclovir, 2'-fluoro-5-iodoaracytosine, and 2'-fluoro-5-methylarauracil but were susceptible to vidarabine or phosphonoformate. These findings confirm that the selective antiviral activity of 5-ethyl-2'-deoxyuridine is mediated by the virus-induced thymidine kinase. Oral or intraperitoneal administration of the drug at nontoxic doses was ineffective in protecting mice against intracerebral challenge with virus. Using implanted osmotic minipumps or coadministering the drug with dimethyl sulfoxide failed to decrease the mortality rate. In guinea pigs infected genitaily with HSV-2, topical drug treatment was more effective than placebo in reducing lesion severity and other clinical and virological variables. These effects were noted whether the drug treatment was initiated 3 or 24 h after infection (ascertained serologically). Drug-treated animals had a significantly lower herpes antibody titer than did placebo-treated guinea pigs, suggesting that the drug can also reduce the viral antigen load. In this model, the drug appeared to be as effective as topical phosphonoformate or acyclovir.5-Ethyl-2'-deoxyuridine (EdU) is a pyrimidine nucleoside with activity in cell culture against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and vaccinia virus (9,16,28,48). The compound is also active in rabbits with herpes keratitis (13, 15), in mice with systemic herpetic infection (7,8), and in dorsally infected guinea pigs (46). Studies in humans have indicated positive effects with this drug for the treatment of ocular herpes (13, 35). EdU does not appear to interfere with regeneration of the epithelium of the rabbit eye (17). EdU has no demonstrable mutagenicity, and the drug does not induce RNA retroviruses in several cell culture systems (20,29,45,48). In rats, more than 90% of the drug or its metabolites are excreted in the kidney within 24 h (24).Since the full spectrum of activity of this drug against HSV is incomplete, several studies were initiated which had the following objectives: (i) delineating the activity of EdU against 2 laboratory strains and 12 clinical isolates of HSV-1 and 1 laboratory strain and 12 clinical isolates of HSV-2; (ii) comparing the cell culture activity of EdU with those of acyclovir (ACV), vidarabine (ara-A), E-5-(2-bromovinyl)-2'-deoxyuridine, 2'-fluoro-5-methylarauracil (FMAU), and 2'-fluoro-5-iodoaracytosine against laboratory strains of HSV; (iii) assessing activities of several of these agents against EdU-resistant variants; (iv) determining the capacity of EdU administered ...

show abstract

Specific phosphorylation of 5-ethyl-2′-deoxyuridine by herpes simplex virus-infected cells and incorporation into viral DNA.

Clercq

Bernaerts

1987

Journal of Biological Chemistry

View full text Add to dashboard Cite

Differential Substrate Specificity of DNA Polymerase beta and of a DNA Polymerase Induced by Herpes Simplex Virus Type 2 towards Thymidine Triphosphate Analogues

Cited by 5 publications

References 18 publications

Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

Antiviral activity of 5-ethyl-2'-deoxyuridine against herpes simplex viruses in cell culture, mice, and guinea pigs

Specific phosphorylation of 5-ethyl-2′-deoxyuridine by herpes simplex virus-infected cells and incorporation into viral DNA.

Contact Info

Product

Resources

About