DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

Abstract

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphatebinding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-␤-D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early-and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.Vaccinia virus (VV), the prototype poxvirus, replicates within the cytoplasm and has a linear double-stranded DNA genome of 185 kbp with inverted terminal repeats (16, 50) and covalently linked ends (2,17). Approximately 185 open reading frames (ORFs) are likely to encode proteins, though the functions of less than half of these are known (21,36). Some insights into protein function have come from comparative sequence analysis. Eight VV ORFs (A18, A32, A48, D5, D6, D11, I8, and J2) have predicted purine nucleoside triphosphate binding motifs (18,27,28), and there is evidence that most of the corresponding proteins have roles in transcription, replication, alteration of DNA topology, or nucleotide metabolism. Of these proteins, least is known about the A32 gene product. Based on limited sequence similarity to the products of gene I of filamentous, single-stranded DNA bacteriophages and to the Iva2 gene of adenovirus, Koonin et al. (28) suggested that the A32 gene product may be an ATPase involved in DNA packaging. In addition, a highly conserved function was predicted from the degree of similarity of the deduced amino acid sequences of the VV A32 protein to its homolog in the distantly related molluscum contagiosum virus (43,44).In the absence of any experimental data regarding the expression or role of the VV A32 gene, we have taken an in vivo genetic approach to the subject. Here we describe the construction and properties of a conditional lethal mutant of VV with an inducible A32 gene. MATERIALS AND METHODS Cells and viruses. BS-C-1 (ATCC CCL6) and HeLa S3 cells (ATCC C...

Vaccinia Virus WR Gene A5L Is Required for Morphogenesis of Mature Virions

“…Since the protein coding sequence for A5 was identical to the sequence of WR, the optimal virus production at the elevated level of A5 reflects a less efficient utilization of the protein, either directly or by some posttranslational process in the cells infected with the recombinant virus. Similar elevated levels of inducer gene expression for optimum virus production has been observed previously by using the VOTE expression system (5,19,54). At the inducer concentration of 250 M IPTG, even though a higher level of A5 production was observed (data not shown), a decrease in vA5Li production was noted (Fig.…”

“…This T7 RNA polymerase inducible system was designed to scale the level of expression of the target gene to the concentration of IPTG. This property allows one to express the target gene at the optimum level, as overexpression of the target gene is sometimes deleterious to viral growth (5). The relationship between inducer concentration and vA5Li virus yield was determined by plaque assay of infected cells as illustrated in Fig.…”

“…Three basic approaches have been used to delineate the molecular details of vaccinia virus morphogenesis: the characterization of temperature-sensitive mutants with defects in assembly, the use of drugs which inhibit the process and the characterization of mutant viruses resistant to such drugs, and the construction of inducer-dependent conditional mutants in which the expression of a specified gene can be controlled. By using these approaches, proteins directly or indirectly involved in the early stages of morphogenesis (36,37,46,47,51,54,55), in the transition from IV to IMV particles (5,13,22,35,57), and in the process leading to the formation of extracellular enveloped virus (3,9,12,34,38,39,42,53) have been identified.…”

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

“…Thus, the core protein VP8 (L4R) is thought to be required for correct packaging of the viral genome and/or for the efficient transcription of DNA (61), whereas DNA-binding phosphoprotein VP11 (F18R) (67) and the I7 protein, homologous to yeast type II DNA topoisomerase (24), appear to be essential for nucleoid condensation. The A32 protein appears to be required at an earlier step, since repression of A32 results in the accumulation of DNA-deficient IVs (4). Also other DNAbinding proteins, such as the VV early transcription factor VETF, composed of two subunits encoded by the D6R and A8L genes, and the product of the I1L gene are needed for IV-to-IMV transition since viruses deficient in these polypeptides are blocked at the IV stage (19,20,27,29).…”

Genome sequence of tsetse bracoviruses: insights into symbiotic virus evolution

“…In this work, we identify and use transposable elements designated Mavericks (Herniou et al 2013) to underpin the genetic diversity of PDVs in the recently sequenced Dipteran genomes. Our Maverick sequences consist of four genes: a DNA polymerase B2 involved in DNA excision repair and initiation of replication (DNA Pol B 2) (Drezen et al 2006), the N terminal region of the parvovirus coat protein VP1 (Parvo coat N) that is important for virion retention and transduction by insect vectors (Spitzer et al 1997); a retroviral-like integrase; and poxvirus A32 protein (Pox A32) that encodes an ATPase involved in virion DNA packaging (Cassetti et al 1998). Maverick associated genes have been shown to occur in close proximity to each other, and are thought to be genetically linked (Dupuy et al 2011).…”