scholarly journals Genotypic Characterization of the DNA Polymerase and Sensitivity to Antiviral Compounds of Foscarnet-Resistant Herpes Simplex Virus Type 1 (HSV-1) Derived from a Foscarnet-Sensitive HSV-1 Strain

2005 ◽  
Vol 49 (2) ◽  
pp. 606-611 ◽  
Author(s):  
Masayuki Saijo ◽  
Tatsuo Suzutani ◽  
Shigeru Morikawa ◽  
Ichiro Kurane
Keyword(s):  
Amino Acid ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Amino Acid Substitution ◽  
Single Amino Acid ◽  
Conserved Region ◽  
Simplex Virus ◽  
Region Ii ◽  
Hsv 1

ABSTRACT Foscarnet is widely used for the treatment of acyclovir-resistant herpesvirus infections, and foscarnet-resistant herpesvirus infections are a serious concern in immunocompromised patients. Twenty-seven single-plaque isolates of herpes simplex virus type 1 (HSV-1) resistant to foscarnet were selected from foscarnet- and acyclovir-sensitive HSV-1 strain TAS by exposure to foscarnet, and the DNA polymerase genes were analyzed. The sensitivities of these mutants to foscarnet, cidofovir, S2242, acyclovir, ganciclovir, and penciclovir were determined. A single amino acid substitution, double amino acid substitutions, and a combination of a single amino acid substitution with a deletion or insertion of amino acid residues in the viral DNA polymerase were demonstrated in 21, 4, and 2 isolates, respectively. Of the 27 isolates, an amino acid substitution of serine for asparagine at amino acid position 724 in the DNA polymerase (724 S-N) was detected in 8 isolates. An amino acid substitution in conserved region II was demonstrated in these eight isolates as well as four other isolates. The mutation in the DNA polymerase responsible for resistance to foscarnet was located between the pre-IV region and conserved region V, especially within conserved region II. All the isolates were sensitive or hypersensitive to cidofovir and ganciclovir. Seven, 5, and 15 of the 27 isolates were also sensitive to S2242, acyclovir, and penciclovir, respectively. Thus, most of the foscarnet-resistant HSV-1 isolates were sensitive or hypersensitive to cidofovir and ganciclovir.

scholarly journals Amino Acid Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus Antiviral Agents

2003 ◽  
Vol 77 (3) ◽  
pp. 1868-1876 ◽  
Author(s):  
Darrell R. Thomsen ◽  
Nancee L. Oien ◽  
Todd A. Hopkins ◽  
Mary L. Knechtel ◽  
Roger J. Brideau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Conserved Domain ◽  
Domain Iii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.

scholarly journals 3′ to 5′ Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase Modulates Its Strand Displacement Activity

2003 ◽  
Vol 77 (18) ◽  
pp. 10147-10153 ◽  
Author(s):  
Yali Zhu ◽  
Kelly S. Trego ◽  
Liping Song ◽  
Deborah S. Parris
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Strand Displacement ◽  
Displacement Activity ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant strand displacement activity with or without its processivity factor, UL42. However, an exonuclease-deficient (exo−) pol (D368A) was capable of slow strand displacement. Although UL42 increased the rate (2/s) and processivity of strand displacement by exo− pol, the rate was slower than that for gap-filling synthesis. High inherent excision rates on matched primer-templates and rapid idling-turnover (successive rounds of excision and polymerization) of exo-proficient polymerases correlated with poor strand displacement activity. The results suggest that the exo activity of HSV-1 pol modulates its ability to engage in strand displacement, a function that may be important to the viability and genome stability of the virus.

scholarly journals Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance

2015 ◽  
Vol 59 (8) ◽  
pp. 4938-4945 ◽  
Author(s):  
Susanne Schmidt ◽  
Kathrin Bohn-Wippert ◽  
Peter Schlattmann ◽  
Roland Zell ◽  
Andreas Sauerbrei
Keyword(s):  
Herpes Simplex Virus ◽  
Amino Acid ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Dna Polymerase ◽  
Herpes Simplex Virus 1 ◽  
Immunosuppressed Patients ◽  
Resistant Strains ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTA total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance.

scholarly journals Antiviral activity of the 3′;-amino derivative of (E)-5-(2-bromovinyl)-2′;-deoxyuridine

1983 ◽  
Vol 211 (2) ◽  
pp. 439-445 ◽  
Author(s):  
E De Clercq ◽  
J Descamps ◽  
J Balzarini ◽  
T Fukui ◽  
H S Allaudeen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Amino Derivative ◽  
Dna Polymerase Alpha ◽  
Simplex Virus ◽  
Hsv 1

3′-NH2-BV-dUrd, the 3′-amino derivative of (E)-5-(2-bromovinyl)-2′-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3′-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3′-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5′-triphosphate of 3′-NH2-BV-dUrd (3′-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3′-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3′-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5′-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3′;-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.

scholarly journals Evidence of Muller’s ratchet in herpes simplex virus type 1

2013 ◽  
Vol 94 (2) ◽  
pp. 366-375 ◽  
Author(s):  
Nacarí Jaramillo ◽  
Esteban Domingo ◽  
María Carmen Muñoz-Egea ◽  
Enrique Tabarés ◽  
Ignacio Gadea
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Herpes Simplex Virus Type ◽  
Rna Viruses ◽  
Population Bottlenecks ◽  
Dna Viruses ◽  
Simplex Virus ◽  
Hsv 1

Population bottlenecks can have major effects in the evolution of RNA viruses, but their possible influence in the evolution of DNA viruses is largely unknown. Genetic and biological variation of herpes simplex virus type 1 (HSV-1) has been studied by subjecting 23 biological clones of the virus to 10 plaque-to-plaque transfers. In contrast to large population passages, plaque transfers led to a decrease in replicative capacity of HSV-1. Two out of a total of 23 clones did not survive to the last transfer in 143 TK– cells. DNA from three genomic regions (DNA polymerase, glycoprotein gD and thymidine kinase) from the initial and passaged clones was sequenced. Nucleotide substitutions were detected in the TK and gD genes, but not in the DNA polymerase gene. Assuming a uniform distribution of mutations along the genome, the average rate of fixation of mutations was about five mutations per viral genome and plaque transfer. This value is comparable to the range of values calculated for RNA viruses. Four plaque-transferred populations lost neurovirulence for mice, as compared with the corresponding initial clones. LD50 values obtained with the populations subjected to serial bottlenecks were 4- to 67-fold higher than for their parental clones. These results equate HSV-1 with RNA viruses regarding fitness decrease as a result of plaque–to-plaque transfers, and show that population bottlenecks can modify the pathogenic potential of HSV-1. Implications for the evolution of complex DNA viruses are discussed.

scholarly journals The Ig-Like V-Type Domain of Paired Ig-Like Type 2 Receptor Alpha Is Critical for Herpes Simplex Virus Type 1-Mediated Membrane Fusion

2010 ◽  
Vol 84 (17) ◽  
pp. 8664-8672 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker
Keyword(s):  
Amino Acid ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Fusion Activity ◽  
Acid Protein ◽  
Simplex Virus ◽  
Amino Acid Protein ◽  
Hsv 1

ABSTRACT Paired immunoglobulin (Ig)-like type 2 receptor alpha (PILRα) and PILRβ are paired receptors that are highly homologous to each other. When engaged by ligand, PILRα is inhibitory whereas PILRβ is activating. PILRα is a newly identified herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) receptor and is associated with membrane fusion and entry activity of HSV-1. PILRα is a 303-amino-acid protein with an Ig-like V (variable)-type domain from amino acid 31 to 150, whereas PILRβ is a 217-amino-acid protein with an Ig-like V-type domain from amino acid 21 to 143. We report that PILRβ is not a receptor for HSV-1 and HSV-2. Domain swaps between PILRα and PILRβ reveal that the Ig-like V-type domain of PILRα, but not PILRβ, plays a critical role in cell membrane fusion activity and the binding of PILRα to gB. Individual replacement of 13 amino acids in PILRα showed that most of these mutations had no effect on cell fusion activity. However, mutation of the tryptophan residue at amino acid 139 significantly impaired cell fusion activity for HSV-1 and eliminated binding to gB.

scholarly journals Kinetic Approaches to Understanding the Mechanisms of Fidelity of the Herpes Simplex Virus Type 1 DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
Author(s):  
Yali Zhu ◽  
Jason Stroud ◽  
Liping Song ◽  
Deborah S. Parris
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Active Sites ◽  
Replicative Polymerases ◽  
Simplex Virus ◽  
Hsv 1

We discuss how the results of presteady-state and steady-state kinetic analysis of the polymerizing and excision activities of herpes simplex virus type 1 (HSV-1) DNA polymerase have led to a better understanding of the mechanisms controlling fidelity of this important model replication polymerase. Despite a poorer misincorporation frequency compared to other replicative polymerases with intrinsic 3′to 5′exonuclease (exo) activity, HSV-1 DNA replication fidelity is enhanced by a high kinetic barrier to extending a primer/template containing a mismatch or abasic lesion and by the dynamic ability of the polymerase to switch the primer terminus between the exo and polymerizing active sites. The HSV-1 polymerase with a catalytically inactivated exo activity possesses reduced rates of primer switching and fails to support productive replication, suggesting a novel means to target polymerase for replication inhibition.

scholarly journals Samarangenin B from Limonium sinense Suppresses Herpes Simplex Virus Type 1 Replication in Vero Cells by Regulation of Viral Macromolecular Synthesis

2002 ◽  
Vol 46 (9) ◽  
pp. 2854-2864 ◽  
Author(s):  
Yuh-Chi Kuo ◽  
Lie-Chwen Lin ◽  
Wei-Jern Tsai ◽  
Cheng-Jen Chou ◽  
Szu-Hao Kung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Herpes Simplex Virus Type ◽  
Vero Cells ◽  
Limonium Sinense ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, samarangenin B (Sam B) was isolated from Limonium sinense; Sam B significantly suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of Sam B on HSV-1 replication was not due to the blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral immediate-early (α), early (β), and late (γ) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB), gC, gD, gG, and infected-cell protein 5 (ICP5) expression and gB mRNA expression in Vero cells were impeded by Sam B. Data from PCR showed that replication of HSV-1 DNA in Vero cells was arrested by Sam B. Furthermore, Sam B decreased DNA polymerase, ICP0, and ICP4 gene expression in Vero cells. Results of an electrophoretic mobility shift assay demonstrated that Sam B interrupted the formation of an α-trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of Sam B seem to be mediated, at least in part, by inhibiting HSV-1 α gene expression, including expression of the ICP0 and ICP4 genes, by blocking β transcripts such as DNA polymerase mRNA, and by arresting HSV-1 DNA synthesis and structural protein expression in Vero cells. These results show that Sam B is an antiviral agent against HSV-1 replication.

scholarly journals Herpes Simplex Virus Type 1 DNA Polymerase Requires the Mammalian Chaperone Hsp90 for Proper Localization to the Nucleus

2005 ◽  
Vol 79 (16) ◽  
pp. 10740-10749 ◽  
Author(s):  
April D. Burch ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Hsp90 Chaperone ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many viruses and bacteriophage utilize chaperone systems for DNA replication and viral morphogenesis. We have previously shown that in the herpes simplex virus type 1 (HSV-1)-infected cell nucleus, foci enriched in the Hsp70/Hsp40 chaperone machinery are formed adjacent to viral replication compartments (A. D. Burch and S. K. Weller, J. Virol. 78:7175-7185, 2004). These foci have now been named virus-induced chaperone-enriched (VICE) foci. Since the Hsp90 chaperone machinery is known to engage the Hsp70/Hsp40 system in eukaryotes, the subcellular localization of Hsp90 in HSV-1-infected cells was analyzed. Hsp90 is found within viral replication compartments as well as in the Hsp70/Hsp40-enriched foci. Geldanamycin, an inhibitor of Hsp90, results in decreased HSV-1 yields and blocks viral DNA synthesis. Furthermore, we have found that the viral DNA polymerase is mislocalized to the cytoplasm in both infected and transfected cells in the presence of geldanamycin. Additionally, in the presence of an Hsp90 inhibitor, proteasome-dependent degradation of the viral polymerase was detected by Western blot analysis. These data identify the HSV-1 polymerase as a putative client protein of the Hsp90 chaperone system. Perturbations in this association appear to result in degradation, aberrant folding, and/or intracellular localization of the viral polymerase.

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