scholarly journals Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

2006 ◽  
Vol 81 (3) ◽  
pp. 1433-1443 ◽  
Author(s):  
Xiangzhi Meng ◽  
Addie Embry ◽  
Debbi Sochia ◽  
Yan Xiang
Keyword(s):  
Vaccinia Virus ◽  
Core Protein ◽  
Viral Gene Expression ◽  
Recombinant Vaccinia Virus ◽  
Proteolytic Processing ◽  
Viral Gene ◽  
Temperature Sensitive ◽  
Virion Morphogenesis ◽  
Viral Factory ◽  
Virion Core

ABSTRACT Vaccinia virus A6L is a previously uncharacterized gene that is conserved in all sequenced vertebrate poxviruses. Here, we constructed a recombinant vaccinia virus encoding A6 with an epitope tag and showed that A6 was expressed in infected cells after viral DNA replication and packaged in the core of the mature virion. Furthermore, we showed that A6 was essential for vaccinia virus replication by performing clustered charge-to-alanine mutagenesis on A6, which resulted in two vaccinia virus mutants (vA6L-mut1 and vA6L-mut2) that displayed a temperature-sensitive phenotype. At 31°C, both mutants replicated efficiently; however, at 40°C, vA6L-mut1 grew to a low titer, while vA6L-mut2 failed to replicate. The A6 protein expressed by vA6L-mut2 exhibited temperature-dependent instability. At the nonpermissive temperature, vA6L-mut2 was normal at viral gene expression and viral factory formation, but it was defective for proteolytic processing of the precursors of several major virion proteins, a defect that is characteristic of a block in virion morphogenesis. Electron microscopy further showed that the morphogenesis of vA6L-mut2 was arrested before the formation of immature virion with nucleoid and mature virion. Taken together, our data show that A6 is a virion core protein that plays an essential role in virion morphogenesis.

scholarly journals Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus H5 Gene: Isolation of a Dominant, Temperature-Sensitive Mutant with a Profound Defect in Morphogenesis

2000 ◽  
Vol 74 (5) ◽  
pp. 2393-2405 ◽  
Author(s):  
Joseph DeMasi ◽  
Paula Traktman
Keyword(s):  
Gene Expression ◽  
Vaccinia Virus ◽  
Viral Gene Expression ◽  
Gene Isolation ◽  
Viral Gene ◽  
Temperature Sensitive ◽  
Genome Replication ◽  
Nonpermissive Temperature ◽  
Viral Yield ◽  
Virion Morphogenesis

ABSTRACT The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expressed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcription and, as a substrate of the B1 kinase, may participate in genome replication. To enable a genetic analysis of the role of H5 during the viral life cycle, we used clustered charge-to-alanine mutagenesis in an attempt to create a temperature-sensitive (ts) virus with a lesion in the H5 gene. Five mutant viruses were isolated, with one of them,tsH5-4, having a strong ts phenotype as assayed by plaque formation and measurements of 24-h viral yield. Surprisingly, no defects in genome replication or viral gene expression were detected at the nonpermissive temperature. By electron microscopy, we observed a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature particles. Nonfunctional, “curdled” virosomes were detected in tsH5-4 infections at the nonpermissive temperature. These structures appeared to revert to functional virosomes after a temperature shift to permissive conditions. We suggest an essential role for H5 in normal virosome formation and the initiation of virion morphogenesis. By constructing recombinant genomes containing two H5 alleles, wild type and H5-4, we determined that H5-4 exerted a dominant phenotype. tsH5-4 is the first example of a dominant ts mutant isolated and characterized in vaccinia virus.

scholarly journals The Vaccinia Virus A9L Gene Encodes a Membrane Protein Required for an Early Step in Virion Morphogenesis

2000 ◽  
Vol 74 (20) ◽  
pp. 9701-9711 ◽  
Author(s):  
Wendy W. Yeh ◽  
Bernard Moss ◽  
Elizabeth J. Wolffe
Keyword(s):  
Vaccinia Virus ◽  
Early Stage ◽  
Protein A ◽  
Recombinant Vaccinia Virus ◽  
Proteolytic Processing ◽  
Transcriptional Analysis ◽  
Recombinant Vaccinia ◽  
Reading Frame ◽  
Virus Particles ◽  
Virion Morphogenesis

ABSTRACT The A9L open reading frame of vaccinia virus was predicted to encode a membrane-associated protein. A transcriptional analysis of the A9L gene indicated that it was expressed at late times in vaccinia virus-infected cells. Late expression, as well as virion membrane association, was demonstrated by the construction and use of a recombinant vaccinia virus encoding an A9L protein with a C-terminal epitope tag. Immunoelectron microscopy revealed that the A9L protein was associated with both immature and mature virus particles and was oriented in the membrane with its C terminus exposed on the virion surface. To determine whether the A9L protein functions in viral assembly or infectivity, we made a conditional-lethal inducible recombinant vaccinia virus. In the absence of inducer, A9L expression and virus replication were undetectable. Under nonpermissive conditions, viral late protein synthesis occurred, but maturational proteolytic processing was inhibited, and there was an accumulation of membrane-coated electron-dense bodies, crescents, and immature virus particles, many of which appeared abnormal. We concluded that the product of the A9L gene is a viral membrane-associated protein and functions at an early stage in virion morphogenesis.

scholarly journals Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

2004 ◽  
Vol 78 (12) ◽  
pp. 6335-6343 ◽  
Author(s):  
Camilo Ansarah-Sobrinho ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Lethal Mutant ◽  
Core Proteins ◽  
Core Components

ABSTRACT Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.

scholarly journals Vaccinia Virus Mutants with Alanine Substitutions in the Conserved G5R Gene Fail To Initiate Morphogenesis at the Nonpermissive Temperature

2004 ◽  
Vol 78 (19) ◽  
pp. 10238-10248 ◽  
Author(s):  
Flavio G. da Fonseca ◽  
Andrea S. Weisberg ◽  
Maria F. Caeiro ◽  
Bernard Moss
Keyword(s):  
Dna Replication ◽  
Vaccinia Virus ◽  
Viral Gene Expression ◽  
Proteolytic Cleavage ◽  
Viral Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Reading Frame ◽  
Charged Amino Acids ◽  
Core Proteins

ABSTRACT The initial characterization of the product of the vaccinia virus G5R gene, which is conserved in all poxviruses sequenced to date, is described. The G5 protein was detected in the core fraction of purified virions, and transcription and translation of the G5R open reading frame occurred early in infection, independently of DNA replication. Attempts to delete the G5R gene and isolate a replication-competent virus were unsuccessful, suggesting that G5R encodes an essential function. We engineered vaccinia virus mutants with clusters of charged amino acids changed to alanines and determined that several were unable to replicate at 40°C but grew well at 37°C. At the nonpermissive temperature, viral gene expression and DNA replication and processing were unperturbed. However, tyrosine phosphorylation and proteolytic cleavage of the A17 membrane protein and proteolytic cleavage of core proteins were inhibited at 40°C, suggesting an assembly defect. The cytoplasm of cells that had been infected at the nonpermissive temperature contained large granular areas devoid of cellular organelles or virus structures except for occasional short crescent-shaped membranes and electron-dense lacy structures. The temperature-sensitive phenotype of the G5R mutants closely resembled the phenotypes of vaccinia virus mutants carrying conditionally lethal F10R protein kinase and H5R mutations. F10, although required for phosphorylation of A17 and viral membrane formation, was synthesized by the G5R mutants under nonpermissive conditions. An intriguing possibility is that G5 participates in the formation of viral membranes, a poorly understood event in poxvirus assembly.

scholarly journals Vaccinia Virus Nonstructural Protein Encoded by the A11R Gene Is Required for Formation of the Virion Membrane

2005 ◽  
Vol 79 (11) ◽  
pp. 6598-6609 ◽  
Author(s):  
Wolfgang Resch ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Posttranslational Modifications ◽  
Nonstructural Protein ◽  
Recombinant Vaccinia Virus ◽  
Dna Packaging ◽  
Proteolytic Processing ◽  
Particle Assembly ◽  
Cycle Growth ◽  
Infected Cells ◽  
Viral Factory

ABSTRACT The vaccinia virus A11R gene has orthologs in all known poxvirus genomes, and the A11 protein has been previously reported to interact with the putative DNA packaging protein A32 in a yeast two-hybrid screen. Using antisera raised against A11 peptides, we show that the A11 protein was (i) expressed at late times with an apparent mass of 40 kDa, (ii) not incorporated into virus particles, (iii) phosphorylated independently of the viral F10 kinase, (iv) coimmunoprecipitated with A32, and (v) localized to the viral factory. To determine the role of the A11 protein and test whether it is indeed involved in DNA packaging, we constructed a recombinant vaccinia virus with an inducible A11R gene. This recombinant was dependent on inducer for single-cycle growth and plaque formation. In the absence of inducer, viral late proteins were produced at normal levels, but proteolytic processing and other posttranslational modifications of some proteins were inhibited, suggesting a block in virus particle assembly. Consistent with this observation, electron microscopy of cells infected in the absence of inducer showed virus factories with abnormal electron-dense viroplasms and intermediate density regions associated with membranes and containing the D13 protein. However, no viral membrane crescents, immature virions, or mature virions were produced. The requirement for nonvirion protein A11 in order to make normal viral membranes was an unexpected and exciting finding, since neither the origin of these membranes nor their mechanism of formation in the cytoplasm of infected cells is understood.

Molecular biology of rubella virus structural proteins

1994 ◽  
Vol 72 (9-10) ◽  
pp. 349-356 ◽  
Author(s):  
Shirley Gillam
Keyword(s):  
Molecular Biology ◽  
Rubella Virus ◽  
Cell Biology ◽  
Intracellular Transport ◽  
Rna Virus ◽  
Viral Gene Expression ◽  
Proteolytic Processing ◽  
Structural Proteins ◽  
Viral Gene ◽  
Structural Protein

Rubella virus is a small, enveloped, positive-stranded RNA virus in the Togaviridae family and bears similarities to the prototype alphaviruses in terms of its genome organization and strategy for viral gene expression. Despite being an important human pathogen, the cell biology of rubella virus remains poorly characterized. This review focuses on the molecular biology of rubella virus structural proteins, with emphasis on the proteolytic processing and maturation of virus structural proteins, the glycosylation requirement for intracellular transport and function of glycoproteins, and the localization of hemagglutinin- and virus-neutralizing epitopes. A number of significant differences between rubella virus and alphavirus structural protein expression and maturation were discovered.Key words: rubella virus, N-linked glycosylation, epitope mapping, proteolytic processing.

scholarly journals Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly

2007 ◽  
Vol 81 (24) ◽  
pp. 13392-13402 ◽  
Author(s):  
Liang Deng ◽  
Peihong Dai ◽  
Anthony Ciro ◽  
Donald F. Smee ◽  
Hakim Djaballah ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Viral Gene Expression ◽  
Maximum Tolerated Dose ◽  
Structural Proteins ◽  
Dna Ligase ◽  
Viral Gene ◽  
Whole Genome ◽  
Missense Mutations ◽  
Virion Assembly

ABSTRACT The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.

scholarly journals Genetic Analysis of the Vaccinia Virus I6 Telomere-Binding Protein Uncovers a Key Role in Genome Encapsidation

2003 ◽  
Vol 77 (20) ◽  
pp. 10929-10942 ◽  
Author(s):  
Olivera Grubisha ◽  
Paula Traktman
Keyword(s):  
Vaccinia Virus ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Recombinant Viruses ◽  
Telomere Binding Protein ◽  
Microscopic Studies ◽  
Infected Cells ◽  
Genome Encapsidation

ABSTRACT The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.

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