scholarly journals The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

2003 ◽  
Vol 77 (10) ◽  
pp. 6014-6028 ◽  
Author(s):  
Sonja Welsch ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Endoplasmic Reticulum ◽  
Dna Replication ◽  
Vaccinia Virus ◽  
Gene Product ◽  
Close Association ◽  
Viral Dna ◽  
Mrna Accumulation ◽  
Double Labeling ◽  
Viral Dna Replication ◽  
Early Protein

ABSTRACT The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogenous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

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 Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

2009 ◽  
Vol 84 (6) ◽  
pp. 3079-3093 ◽  
Author(s):  
Karen Tran ◽  
Jeffrey A. Mahr ◽  
Deborah H. Spector
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Proteasome Activity ◽  
Late Gene ◽  
Viral Dna ◽  
Rna Transcription ◽  
Viral Dna Replication ◽  
Early Protein

ABSTRACT We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.

scholarly journals Regulation of Viral Intermediate Gene Expression by the Vaccinia Virus B1 Protein Kinase

2001 ◽  
Vol 75 (9) ◽  
pp. 4048-4055 ◽  
Author(s):  
Gerald R. Kovacs ◽  
Nikos Vasilakis ◽  
Bernard Moss
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Protein Kinase ◽  
Vaccinia Virus ◽  
Gene Transcription ◽  
Late Gene ◽  
Nonpermissive Temperature ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants (ts2 andts25) that map to B1 fail to efficiently replicate viral DNA. Our goal was to extend studies on the function of B1 by determining if the kinase is required for intermediate or late gene expression, two events that ordinarily depend on viral DNA replication. First, we established that early viral gene expression occurred at the nonpermissive temperature. By using a transfection procedure that circumvents the viral DNA replication requirement, we found that reporter genes regulated by an intermediate promoter were transcribed only under conditions permissive for expression of active B1. To assay late gene expression, the T7 RNA polymerase gene was inserted into the genome of ts25 to form ts25/T7. A DNA replication-independent late gene transcription system was established by cotransfecting plasmids containing T7 promoter-driven late gene transcription factors and a late promoter reporter gene intots25/T7-infected cells. Late genes, unlike intermediate genes, were expressed at the nonpermissive temperature. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in cells infected with wild-type virus.

scholarly journals Biochemical and Genetic Analysis of the Vaccinia Virus D5 Protein: Multimerization-Dependent ATPase Activity Is Required To Support Viral DNA Replication

2006 ◽  
Vol 81 (2) ◽  
pp. 844-859 ◽  
Author(s):  
Kathleen A. Boyle ◽  
Lisa Arps ◽  
Paula Traktman
Keyword(s):  
Dna Replication ◽  
Enzymatic Activity ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Structural Features ◽  
Temperature Sensitive ◽  
Phenotypic Analysis ◽  
Viral Dna ◽  
Catalytic Core ◽  
Viral Dna Replication

ABSTRACT The vaccinia virus-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive (ts) D5 mutants (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7°C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7°C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive Cts24 infections, only a wtD5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA+ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA+ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.

scholarly journals Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells

2015 ◽  
Vol 89 (20) ◽  
pp. 10247-10259 ◽  
Author(s):  
Augusta Jamin ◽  
Nouhou Ibrahim ◽  
April Wicklund ◽  
Kaitlin Weskamp ◽  
Matthew S. Wiebe
Keyword(s):  
Life Cycle ◽  
Dna Replication ◽  
Vaccinia Virus ◽  
Host Defense ◽  
Cell Types ◽  
Viral Life Cycle ◽  
Mutant Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
U2os Cells

ABSTRACTThe vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle.IMPORTANCEThe most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.

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