Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase

Liuzzi, Michel; Kibler, Philip; Bousquet, Christiane; Harji, Fayaz; Bolger, Gordon T.; Garneau, Michel; Lapeyre, Nicole; McCollum, Robert S.; Faucher, Anne‐Marie; Simoneau, Bruno; Cordingley, Michael G.

doi:10.1016/j.antiviral.2004.02.007

Cited by 41 publications

(37 citation statements)

References 26 publications

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“…Alternatively, UL5 and UL52 may share a DNA-binding interface. The appearance of drug resistant mutations for HSV helicase-primase inhibitors in both UL5 and UL52 subunits suggests that inhibitors may bind at the interface between the UL5 and UL52 subunits (4,26,32).…”

Section: Discussionmentioning

confidence: 99%

“…The sequences of the three 60-mer partially cDNA oligonucleotides, 4x12-1, 4x12-3, and 4x12-4 were described previously (17). Oligonucleotide 4x12-4 was 5Ј end labeled using [␥- 32 P]ATP (6,000 Ci/mmol [222 TBq]) and polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled oligonucleotide 4x12-4 was annealed to the appropriate oligonucleotide(s) at a ratio of 1:3 in 50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

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Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

Chen

Carrington-Lawrence

Bai

et al. 2005

J Virol

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Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNAbinding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

Chen

Carrington-Lawrence

Bai

et al. 2005

J Virol

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“…Three mutant viruses were found to have mutations in their UL5 gene [171]. One had Gly352 changed to Val, one had Gly352 changed to Cys, and the third had Lys356 changed to Asn, indicating that the UL5 subunit of the HSV helicaseprimase is likely the target of the BILS inhibitors.…”

Section: Potential Antiviral Agents Sf1 Helicase Inhibitorsmentioning

confidence: 99%

“…DNAstimulated ATP hydrolysis catalyzed by purified UL5/UL52/UL8 complexes with these mutations is less sensitive to the inhibitors. Both Gly352 and Lys356 are completely conserved in all human herpesviruses [172], but the mutant viruses are viable and still cause disease [171]. The frequency with which BILS resistant mutants arise is significantly less than that seen for acyclovir-resistant mutants [171].…”

Section: Potential Antiviral Agents Sf1 Helicase Inhibitorsmentioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

103

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Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

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“…On the other hand, mutations in the UL52 zinc finger motif affect DNA binding of the entire complex, and mutations in UL5 affect primase activity (4,16). Furthermore, mutations causing resistance to helicase-primase inhibitors have been mapped to both UL5 and UL52 subunits, suggesting that interactions of these two subunits create a composite substrate binding surface (2,5,30,36). Recent studies with subcomplexes containing various subsets of the helicase-primase complex subunits suggest that UL52-UL8 complex can polymerize nucleoside triphosphates (NTPs) onto an RNA primer-template, indicating that UL52 contains the active site for phosphodiester bond formation (14).…”

mentioning

confidence: 99%

Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation

Chen

Bai

Mackay

et al. 2011

J Virol

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The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5 to 3 helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.

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Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase

Cited by 41 publications

References 26 publications

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

Understanding Helicases as a Means of Virus Control

Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation

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