Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Section: Discussionmentioning

confidence: 99%

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

Jesus

Costa

Gonçalves

et al. 2009

“…Vaccinia virus IMV particles were extracted with 1% NP-40 containing 50 mM dithiothreitol (DTT) to separate the membrane and core fractions, as described previously (8). In brief, purified IMV (10 8 PFU) was incubated for 1 h at 37°C in 1% NP-40, 50 mM Tris-HCl, pH 7.5, and 50 mM dithiothreitol, and the insoluble and soluble fractions were separated by centrifugation at 14,000 ϫ g for 30 min at 4°C.…”

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confidence: 99%

“…The A26L (WR) PCR DNA fragment was cloned into plasmid pA25-KS(Ϫ) to produce plasmid pA25/A26L(WR)-KS(Ϫ). The 3-kb p11k-lacZ gene expression cassette was isolated from pSC11-5t by SalI and PstI digestion as described previously (8) and ligated with the A25/ A26L(WR)-KS(Ϫ) plasmid to create plasmid A25/A26L(WR)/lacZ-KS(Ϫ). The 3Ј-flanking fragment was generated by PCR using IA27L viral genomic DNA as template and the following two primers (NotI and SmaI restriction sites are underlined): 5Ј-CCCGCGGCCGCGAATGTTGTTTGTTAATG-3Ј and 5Ј-AA ACCCGGGTCATGTCTGGATCCGTCG-3Ј.…”

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confidence: 99%

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Vaccinia Virus 4c (A26L) Protein on Intracellular Mature Virus Binds to the Extracellular Cellular Matrix Laminin

Chiu

Lin²,

Yang

et al. 2007

Self Cite

128

131

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG ؊ sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.

“…The association of the viral crescent membranes with electron-dense material containing internal virion components requires proteins encoded by the A30L, G7L, and J1R ORFs (2,13,15,16). When expression of any of these proteins is repressed, the cytoplasmic viral factory regions contain large numbers of crescent-shaped and circular membranes distinctly separated from masses of electron-dense material.…”

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confidence: 99%

Physical and Functional Interactions between Vaccinia Virus F10 Protein Kinase and Virion Assembly Proteins A30 and G7

Szajner

Weisberg

Moss

2004

An early step in vaccinia virus morphogenesis, the association of crescent membranes with electron-dense granular material, is perturbed when expression of the viral protein encoded by the A30L or G7L open reading frame is repressed. Under these conditions, we found that phosphorylation of the A17 membrane protein, which is mediated by the F10 kinase, was severely reduced. Furthermore, A30 and G7 stimulated F10-dependent phosphorylation of A17 in the absence of other viral late proteins. Evidence for physical interactions between A30, G7, and F10 was obtained by their coimmunoprecipitation with antibody against A30 or F10. In addition, phosphorylation of A30 was dependent on the F10 kinase and autophosphorylation of F10 was stimulated by A30 and G7. Nevertheless, the association of A30, G7, and F10 occurred even with mutated, catalytically inactive forms of F10. Just as A30 and G7 are mutually dependent on each other for stability, F10 was nearly undetectable in the absence of A30 and G7. The reverse is not true, however, as repression of F10 did not diminish A30 or G7. Interaction of F10 with A30 and G7 presumably occurred within the virus factory areas of the cytoplasm, where each was concentrated. F10 localized predominantly in the cortical region of immature virions, beneath the membrane where A17 is located. F10 remained associated with the particulate core fraction of mature virions after treatment with a nonionic detergent and reducing agent. The formation of protein complexes such as the one involving A30, G7, and F10 may be a mechanism for the regulated packaging and processing of virion components.The assembly of vaccinia virus (VV), the prototype poxvirus, occurs within cytoplasmic viral factory areas that appear largely cleared of cellular organelles. Although studied intensively by electron microscopy, little is known about the molecular events involved in viral morphogenesis. Several viral proteins required for early stages of virus assembly have been identified through the characterization of temperature-sensitive and inducible conditional lethal mutants. Few recognizable membrane structures can be discerned in cells infected under nonpermissive conditions with mutants that map to the F10L open reading frame (ORF) (14,18,20). The product of the F10L ORF has been characterized as a dual-specificity protein kinase that phosphorylates tyrosine as well as serine and threonine residues of target proteins (1,3,8,20). Mutated forms of F10, with single amino acid substitutions in the active site, were unable to transcomplement an inducible conditional lethal F10 mutant, confirming the importance of phosphorylation in virion assembly (14). Studies with F10 conditional lethal mutants suggest that the membrane components A17 (1, 3) and A14 (1) are substrates of the kinase. A17 is needed for an early step in viral membrane formation (11, 21) and A14 is needed at a slightly later stage of assembly (12,19). The precursor of mature A17 is phosphorylated at serine, threonine, and tyrosine residues (1, 3) and is c...