scholarly journals Herpes Simplex Virus Type 1 C-Terminal Variants of the Origin Binding Protein (OBP), OBPC-1 and OBPC-2, Cooperatively Regulate Viral DNA Levels In Vitro, and OBPC-2 Affects Mortality in Mice

2007 ◽  
Vol 81 (19) ◽  
pp. 10699-10711 ◽  
Author(s):  
Malen A. Link ◽  
Priscilla A. Schaffer
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Origin Binding Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Two in-frame, C-terminal isoforms of the herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), OBPC-1 and OBPC-2, and a unique C-terminal transcript, UL8.5, are specified by HSV-1 DNA. As the first isoform identified, OBPC-1 was initially assumed to be the product of the UL8.5 transcript. Recent evidence has demonstrated, however, that OBPC-1 is a cathepsin B-mediated cleavage product of OBP, suggesting that OBPC-2 is the product of the UL8.5 transcript. Because both OBPC-1 and -2 contain the majority of the OBP DNA binding domain, we hypothesized that both may be involved in regulating origin-dependent, OBP-mediated viral DNA replication. In this paper, we demonstrate that OBPC-2 is, indeed, the product of the UL8.5 transcript. The translational start site of OBPC-2 was mapped, and a virus (M571A) that does not express this protein efficiently was constructed. Using M571A, we have shown that OBPC-2 is able to bind origin DNA, even though it lacks seven N-terminal amino acid residues of the previously mapped OBP DNA binding domain, resulting in a revision of the limits of the OBP DNA binding domain. Consistent with their proposed roles in regulating viral DNA replication, OBPC-1 and -2 act together to down-regulate viral DNA replication in vitro. During functional studies in vivo, OBPC-2 was identified as a factor that increases mortality in the mouse ocular model of HSV-1 infection.

scholarly journals HIV Integrase Inhibitors Block Replication of Alpha-, Beta-, and Gammaherpesviruses

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Zhipeng Yan ◽  
Kevin F. Bryant ◽  
Sean M. Gregory ◽  
Magdalena Angelova ◽  
David H. Dreyfus ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rnase H ◽  
Cell Protein ◽  
Hiv Integrase ◽  
Integrase Inhibitors ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Hiv Integrase Inhibitors ◽  
Hsv 1

ABSTRACTThe catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activityin vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activityin vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi’s sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses.IMPORTANCEThe herpesviruses cause considerable morbidity and mortality. Nucleoside analogs have served as effective antiviral agents against the herpesviruses, but resistance can arise through viral mutation. Second-line anti-herpes drugs have limitations in terms of pharmacokinetic properties and/or toxicity, so there is a great need for additional drugs for treatment of herpesviral infections. This study showed that the HIV integrase inhibitors also block herpesviral infection, raising the important potential of a new class of anti-herpes drugs and the prospect of drugs that combat both HIV and the herpesviruses.

scholarly journals Studies of the Silencing of Baculovirus DNA Binding Protein

2007 ◽  
Vol 81 (11) ◽  
pp. 6122-6127 ◽  
Author(s):  
Ilja Quadt ◽  
Jan W. M. van Lent ◽  
Dagmar Knebel-Mörsdorf
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Primary Role ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Expression Of Genes ◽  
Replication Sites

ABSTRACT Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.

scholarly journals Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

2018 ◽  
Vol 63 (3) ◽  
Author(s):  
Peng Xu ◽  
Safder S. Ganaie ◽  
Xiaomei Wang ◽  
Zekun Wang ◽  
Steve Kleiboeker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Parvovirus B19 ◽  
Binding Domain ◽  
Endonuclease Activity ◽  
Ns1 Protein ◽  
Viral Dna ◽  
Erythroid Progenitor ◽  
Viral Dna Replication

ABSTRACTHuman parvovirus B19 (B19V), a member of the genusErythroparvovirusof the familyParvoviridae, is a small nonenveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kb with two inverted terminal repeats (ITRs). B19V infection often results in severe hematological disorders and fetal death in humans. B19V replication follows a model of rolling hairpin-dependent DNA replication, in which the large nonstructural protein NS1 introduces a site-specific single-strand nick in the viral DNA replication origins, which locate at the ITRs. NS1 executes endonuclease activity through the N-terminal origin-binding domain. Nicking of the viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here, we developed a fluorophore-basedin vitronicking assay of the replication origin using the origin-binding domain of NS1 and compared it with the radioactivein vitronicking assay. We used both assays to screen a set of small-molecule compounds (n = 96) that have potential antinuclease activity. We found that the fluorophore-basedin vitronicking assay demonstrates sensitivity and specificity values as high as those of the radioactive assay. Among the 96 compounds, we identified 8 which have an inhibition of >80% at 10 µM in both the fluorophore-based and radioactivein vitronicking assays. We further tested 3 compounds that have a flavonoid-like structure and anin vitro50% inhibitory concentration that fell in the range of 1 to 3 µM. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells andex vivo-expanded human erythroid progenitor cells.

scholarly journals Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

2003 ◽  
Vol 77 (13) ◽  
pp. 7467-7476 ◽  
Author(s):  
Susan L. Uprichard ◽  
David M. Knipe
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Dna Binding ◽  
Conformational Changes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.

scholarly journals The UL15 protein of herpes simplex virus type 1 is necessary for the localization of the UL28 and UL33 proteins to viral DNA replication centres

2008 ◽  
Vol 89 (7) ◽  
pp. 1709-1715 ◽  
Author(s):  
Martin R. Higgs ◽  
Valerie G. Preston ◽  
Nigel D. Stow
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Simplex Virus ◽  
Hsv 1

The UL15, UL28 and UL33 proteins of herpes simplex virus type 1 (HSV-1) are thought to comprise a terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. Immunofluorescence studies confirmed that shortly after infection with wild-type HSV-1 these three proteins localize to viral DNA replication compartments within the nucleus, identified by the presence of the single-stranded DNA-binding protein, ICP8. In cells infected with either UL28- or UL33-null mutants, the other two terminase proteins also co-localized with ICP8. In contrast, neither UL28 nor UL33 was detectable in replication compartments following infection with a UL15-null mutant, although Western blot analysis showed they were present in normal amounts in the infected cells. Provision of UL15 in a complementing cell line restored the ability of all three proteins to localize to replication compartments. These data indicate that UL15 plays a key role in localizing the terminase complex to DNA replication compartments, and that it can interact independently with UL28 and UL33.

Analysis of the DNA-Binding Domain of the HSV-1 Origin-Binding Protein

Virology ◽  
1994 ◽  
Vol 198 (1) ◽  
pp. 71-80 ◽  
Author(s):  
D.W. Martin ◽  
R.M. Muñoz ◽  
D. Oliver ◽  
M.A. Subler ◽  
S. Deb
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Origin Binding Protein ◽  
Hsv 1

scholarly journals Mutations That Decrease DNA Binding of the Processivity Factor of the Herpes Simplex Virus DNA Polymerase Reduce Viral Yield, Alter the Kinetics of Viral DNA Replication, and Decrease the Fidelity of DNA Replication

2007 ◽  
Vol 81 (7) ◽  
pp. 3495-3502 ◽  
Author(s):  
Changying Jiang ◽  
Ying T. Hwang ◽  
John C. W. Randell ◽  
Donald M. Coen ◽  
Charles B. C. Hwang
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Dna Binding ◽  
Viral Dna ◽  
Replication Fidelity ◽  
Viral Dna Replication ◽  
Control Virus ◽  
Processivity Factor ◽  
Simplex Virus

ABSTRACT The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

scholarly journals Phosphorylation of the Herpes Simplex Virus Type 1 Origin Binding Protein

2001 ◽  
Vol 75 (2) ◽  
pp. 628-637 ◽  
Author(s):  
Jennifer A. Isler ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Origin Binding Protein ◽  
Infected Cells ◽  
Simplex Virus ◽  
Replication Initiator

ABSTRACT The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.

scholarly journals Cathepsin B Mediates Cleavage of Herpes Simplex Virus Type 1 Origin Binding Protein (OBP) To Yield OBPC-1, and Cleavage Is Dependent upon Viral DNA Replication

2007 ◽  
Vol 81 (17) ◽  
pp. 9175-9182 ◽  
Author(s):  
Malen A. Link ◽  
Laurie A. Silva ◽  
Priscilla A. Schaffer
Keyword(s):  
Dna Replication ◽  
Stage I ◽  
Stage Ii ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Replication Stage ◽  
C Terminus ◽  
Origin Binding Protein ◽  
Simplex Virus

ABSTRACT Although the seven viral proteins required for herpes simplex virus type 1 (HSV-1) DNA replication have been identified, the mechanism by which viral DNA synthesis is regulated is unclear. HSV-1 DNA replication is thought to occur in two stages: origin-dependent DNA replication (stage I) mediated by the origin binding protein (OBP), followed by origin- and OBP-independent DNA replication (stage II). The mechanism that facilitates the switch from stage I to stage II is unknown; however, it must involve the loss of OBP function or OBP itself from the replication initiation complex. Previous studies from this laboratory identified a transcript (UL8.5) and protein (OBPC) that are in frame with and comprise the C terminus of the gene specifying OBP. Because of its DNA binding ability, OBPC has been hypothesized to mediate the switch from stage I to stage II. Here, we identify a second protein (OBPC-2) that is also in frame with the C terminus of OBP but comprises a smaller portion of the protein. We demonstrate that the protein originally identified (OBPC-1) is a cathepsin B-mediated cleavage product of OBP, while OBPC-2 may be the product of the UL8.5 transcript. We further demonstrate that the cleavage of OBP to yield OBPC-1 is dependent upon viral DNA replication. These results suggest that cleavage may be a mechanism by which OBP levels and/or activity are regulated during infection.

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