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

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.

Proteome‐wide analysis of the serological response to vaccinia and smallpox

“…While antibodies to envelope proteins were expected, we were surprised by how many of the immunodominant antigens were non-surface virion proteins or proteins found predominantly in infected cells. A significant component of the response is directed against core proteins (I1, L4, A10), and proteins that assist with viral morphogenesis (D13, A11) [15,16]. A second criterion for determining immunodominance, in addition to frequency of recognition, was array SI.…”

Vaccinia Virus L2 Protein Associates with the Endoplasmic Reticulum near the Growing Edge of Crescent Precursors of Immature Virions and Stabilizes a Subset of Viral Membrane Proteins

“…Studies with conditional lethal mutants have led to the identification of many VACV proteins (A11 [35], A14 [38,54], A17 [37,56], F10 [34,48,53], G5 [9], H5 [12], H7 [39], and L2 [27]) that are required for the formation of crescent membranes. Dense masses of viroplasm and in some cases vesicles or tubules accumulate in the absence of these assembly proteins.…”

Rana grylio virus as a vector for foreign gene expression in fish cells

“…TK gene exists in many large DNA viruses and encodes a cytoplasmic protein as an early gene of RGV . Deletion or insertion mutations of TK gene had no effect on virus replication (Rodríguez et al, 1992;García et al, 1995;Szajner et al, 2001;Resch et al, 2005). Thereby TK gene is suitable for insertion of a promoter-foreign gene and recombinant RGV construction.…”

“…Recombinant virus could be used for studying individual gene function, constructing delivery vehicle for biological control of some species or diseases. To date, recombinant viruses have been well-studied in some large DNA viruses, such as African swine fever virus (ASFV) (García et al, 1995;Oliveros et al, 1999;Rodríguez et al, 1992Rodríguez et al, , 2009Epifano et al, 2006), vaccinia virus (Satheshkumar et al, 2009;Sood et al, 2008;Szajner et al, 2001;Resch et al, 2005), human cytomegalovirus (Marchini et al, 2001;Xu et al, 2004) and so on. However, most of the above researches are based on mammalian DNA virus, and knowledge about recombinant aquatic DNA virus (such as iridovirus) is very limited (Pallister et al, 2007).…”