Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins

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Vaccinia virus is a large enveloped poxvirus with more than 200 genes in its genome. Although many poxvirus genomes have been sequenced, knowledge of the host and viral protein components of the virions remains incomplete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and host proteins in purified vaccinia intracellular mature virions (IMV). Analysis of the proteins in the IMV showed that it contains 75 viral proteins, including structural proteins, enzymes, transcription factors, and predicted viral proteins not known to be expressed or present in the IMV. We also determined the relative abundances of the individual protein components in the IMV. Finally, 23 IMV-associated host proteins were also identified. This study provides the first comprehensive structural analysis of the infectious vaccinia virus IMV.

Section: Resultsmentioning

confidence: 79%

Vaccinia Virus Proteome: Identification of Proteins in Vaccinia Virus Intracellular Mature Virion Particles

Chung

Chen

et al. 2006

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“…The molecular mass of I6 is predicted to be 43.5 kDa, and apart from counterparts in other poxviruses, no significant homology to any other proteins was found by computer analysis. Our laboratory confirmed that recombinant I6 protein specifically rec-ognizes extrahelical bases and binds to the viral hairpins with great specificity and stability (6). The work described herein was designed to elucidate the role of I6 in vivo.…”

mentioning

confidence: 70%

“…The vaccinia virus I6 protein first came to our attention because of its ability to bind to the telomeric hairpins of the viral genome with high specificity and stability; this interaction is dependent upon the extrahelical bases that distinguish these unusual telomeres (6). We hypothesized that this protein would be involved in an important facet of genome metabolism, either in the initiation of DNA synthesis or the encapsidation of viral DNA.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Grubisha

Traktman

2003

Self Cite

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.Vaccinia virus, the prototypic member of the Poxvirus family, contains a 192-kb double-stranded DNA genome encoding approximately 200 proteins involved in DNA replication, gene expression, morphogenesis, and virus-host interaction (reviewed in reference 27). The genome is a linear duplex with covalently closed hairpin termini. These termini exist in two 104-nucleotide (nt) isoforms (termed flip and flop) which are inverted complements of one another (2, 3). The hairpin comprises a 4-nt terminal loop and a 44-bp highly AϩT-rich duplex stem with 12 extrahelical bases, 10 on one strand and 2 on the other. Extrahelical bases in the termini are maintained during the viral life cycle in all poxviruses, although the positions and numbers of extrahelical bases differ.We have hypothesized that the viral telomeres play a role in DNA replication. The current replication model proposes that a nick proximal to the terminus is introduced, exposing a 3Ј hydroxyl group that serves as the primer terminus for the initiation of DNA synthesis (reviewed in reference 35). Evidence supporting this model was obtained by labeling synchronized infections with [ 3 H]thymidine; radiolabel was first incorporated within 200 bp of the telomere region (30, 31). Additional evidence was provided by studies in our laboratory that used a minichromosome replication assay (8). Minichromosomes contain a plasmid-derived stuffer sequence flanked by ...

“…I6 is a telomere-binding protein that recognizes extrahelical bases at the hairpin termini of the VACV genome. A suitable DNA structure is essential for the formation of a complex between telomere-binding proteins and the hairpin termini (16). Therefore, it is plausible to suppose that alterations in DNA structure induced by the incorporation of nucleoside analogues could compromise the interaction of DNA with DNA-binding proteins and the subsequent encapsidation of the viral genome.…”

Section: Discussionmentioning

confidence: 99%

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

Jesus

Costa

Gonçalves

et al. 2009

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.