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 Nuclear localization of herpesvirus proteins: potential role for the cellular framework.

1983 ◽  
Vol 3 (3) ◽  
pp. 315-324 ◽  
Author(s):  
M P Quinlan ◽  
D M Knipe
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Capsid Protein ◽  
Cell Nucleus ◽  
Binding Protein ◽  
Cell Monolayer ◽  
Dna Binding Protein ◽  
Actual State ◽  
Viral Dna ◽  
Viral Dna Replication

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.

Nuclear localization of herpesvirus proteins: potential role for the cellular framework

1983 ◽  
Vol 3 (3) ◽  
pp. 315-324
Author(s):  
M P Quinlan ◽  
D M Knipe
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Capsid Protein ◽  
Cell Nucleus ◽  
Binding Protein ◽  
Cell Monolayer ◽  
Dna Binding Protein ◽  
Actual State ◽  
Viral Dna ◽  
Viral Dna Replication

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1976 ◽  
Vol 11 (2) ◽  
pp. 79-95 ◽  
Author(s):  
A. J. Levine ◽  
P. C. van der Vliet ◽  
B. Rosenwirth ◽  
C. Anderson ◽  
J. Rabek ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Strand ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Early Protein

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 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.

Inhibition of adenovirus early region IV transcription in vitro by a purified viral DNA binding protein

Nature ◽  
1983 ◽  
Vol 302 (5908) ◽  
pp. 545-547 ◽  
Author(s):  
Hiroshi Handa ◽  
Robert E. Kingston ◽  
Phillip A. Sharp
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Viral Dna ◽  
Early Region ◽  
Transcription In Vitro

scholarly journals DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Gonçalo Frouco ◽  
Ferdinando B. Freitas ◽  
João Coelho ◽  
Alexandre Leitão ◽  
Carlos Martins ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Topoisomerase Ii ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Dna Supercoiling ◽  
Fever Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Replication Sites

ABSTRACT African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (−78.3%), and reduced transcription of a late viral gene (−47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

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