Herpes simplex virus DNA polymerase, thymidine kinase and deoxyribonuclease activities in cells infected with wild type, ultraviolet-irradiated and defective virus

1979 ◽  
Vol 62 (3) ◽  
pp. 163-174 ◽  
Author(s):  
Y. Becker ◽  
B. Gutter ◽  
Yaffa Cohen ◽  
N. Chejanovsky ◽  
S. Rabkin ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Dna Polymerase ◽  
Wild Type ◽  
Simplex Virus ◽  
Defective Virus ◽  
In Cells

Gene polymorphism of thymidine kinase and DNA polymerase in clinical strains of herpes simplex virus

2011 ◽  
Vol 16 (7) ◽  
pp. 989-997 ◽  
Author(s):  
Kathrin Bohn ◽  
Roland Zell ◽  
Michael Schacke ◽  
Peter Wutzler ◽  
Andreas Sauerbrei
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Gene Polymorphism ◽  
Thymidine Kinase ◽  
Dna Polymerase ◽  
Clinical Strains ◽  
Simplex Virus

scholarly journals Herpes Simplex Virus 1 DNA Polymerase RNase H Activity Acts in a 3′-to-5′ Direction and Is Dependent on the 3′-to-5′ Exonuclease Active Site

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Jessica L. Lawler ◽  
Purba Mukherjee ◽  
Donald M. Coen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Rnase H ◽  
Exonuclease Activity ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTThe catalytic subunit (Pol) of herpes simplex virus 1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA-DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3′-to-5′ exonuclease of Pol or whether it is a separate activity, possibly acting on 5′ RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3′-to-5′ exonuclease-deficient mutant, D368A Pol, for DNA polymerase activity, 3′-to-5′ exonuclease activity, and RNase H activityin vitro. Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5′ or 3′ RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity but was drastically impaired for 3′-to-5′ exonuclease activity, with no activity detected even at high enzyme-to-DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3′ or 5′ terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA-DNA hairpin template, but only detectably with a 3′ RNA terminus in a 3′-to-5′ direction and at a rate lower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3′-to-5′ exonuclease activity and that this activity prefers DNA degradation over degradation of RNA from RNA-DNA hybrids.IMPORTANCEHerpes simplex virus 1 (HSV-1) is a member of theHerpesviridaefamily of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5′-to-3′ RNase H activity intrinsic to this enzyme that could remove RNA primers from Okazaki fragments has been particularly controversial. In this study, we were unable to identify RNase H activity of HSV-1 DNA polymerase on RNA-DNA hybrids with 5′ RNA termini. We detected RNase H activity on hybrids with 3′ termini, but this was due to the 3′-to-5′ exonuclease. Thus, HSV-1 is unlikely to use this method to remove RNA primers during DNA replication but may use pathways similar to those used in eukaryotic Okazaki fragment maturation.

scholarly journals Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia

2006 ◽  
Vol 87 (12) ◽  
pp. 3495-3502 ◽  
Author(s):  
Shih-Heng Chen ◽  
Yu-Wen Lin ◽  
Anthony Griffiths ◽  
Wen-Yen Huang ◽  
Shun-Hua Chen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Antiviral Drug ◽  
Wild Type Virus ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Viral Genomes ◽  
Simplex Virus

Laboratory strains of herpes simplex virus lacking thymidine kinase (TK) cannot replicate acutely to detectable levels in mouse trigeminal ganglia and do not reactivate from latency. However, many pathogenic clinical isolates that are resistant to the antiviral drug acyclovir are heterogeneous populations of TK-negative (TK−) and TK-positive (TK+) viruses. To recapitulate this in vivo, mice were infected with mixtures of wild-type virus and a recombinant TK− mutant in various ratios. Following co-infection, the replication, number of latent viral genomes and reactivation efficiency of TK+ virus in trigeminal ganglia were reduced in a manner related to the amount of TK− virus in the inoculum. TK+ virus did not always complement the acute replication or increase the number of latent viral genomes of TK− mutant in mouse ganglia. Even so, TK+ virus could still confer the pathogenic phenotype to a TK− mutant, somehow providing sufficient TK activity in trans to permit a TK− mutant to reactivate from latently infected ganglia.

scholarly journals Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

2001 ◽  
Vol 75 (17) ◽  
pp. 7904-7912 ◽  
Author(s):  
Sunil J. Advani ◽  
Ryan Hagglund ◽  
Ralph R. Weichselbaum ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infected Cell ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Made In ◽  
In Cells

ABSTRACT The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.

scholarly journals The Enhanced DNA Replication Fidelity of a Mutant Herpes Simplex Virus Type 1 DNA Polymerase Is Mediated by an Improved Nucleotide Selectivity and Reduced Mismatch Extension Ability

2008 ◽  
Vol 82 (17) ◽  
pp. 8937-8941 ◽  
Author(s):  
Wang Tian ◽  
Ying T. Hwang ◽  
Charles B. C. Hwang
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Replication Fidelity ◽  
Simplex Virus ◽  
Dna Replication Fidelity

ABSTRACT We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.

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