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. 2001 Jul;75(13):5752-61.
doi: 10.1128/JVI.75.13.5752-5761.2001.

Vaccinia virus A30L protein is required for association of viral membranes with dense viroplasm to form immature virions

P Szajner  1 , A S Weisberg, E J Wolffe, B Moss
Affiliations

Vaccinia virus A30L protein is required for association of viral membranes with dense viroplasm to form immature virions

P Szajner et al. J Virol. 2001 Jul.

Abstract

The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.

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Figures

FIG. 1
FIG. 1
Transcriptional analysis of the A30L gene. (A) Schematic diagram of the A30L and adjacent ORFs. Arrows above the ORFs indicate the predicted locations of the promoters and the directions of transcription. The dashed arrows below the ORFs represent RNAs that could be initiated from the A30L or the A32L promoter. The solid bars below the dashed arrows represent the full-length 202-nucleotide (nt) riboprobe and the 112-nucleotide segment of the probe that would be protected from nuclease digestion by hybridization to a mRNA that initiated at the putative A30L promoter. (B) RNase protection assays of A30L and F18R transcripts. At the indicated hours postinfection (hpi), total RNA was extracted from uninfected cells or from VV-infected cells incubated for various times in the absence or for 8 h in the presence of cycloheximide (Cx). The polyadenylylated RNA was purified from each sample and hybridized to uniformly 32P-labeled RNA probes complementary to the 5′ ends and upstream sequences of the A30L or F18R ORFs. Hybridized samples were digested with a mixture of RNase T1, RNase A, and S1 nuclease, and the remaining probe fragments were analyzed by PAGE and autoradiography. The numbers on the left represent the sizes in nucleotides of 32P-labeled RNA markers (RNA Century; Ambion). Lane P contained full-length undigested probe; the remaining lanes contained samples derived from cells mock infected for 8 h (U) or from cells harvested at 2 through 24 h after infection. The expected positions of the probe fragments protected by the A30L and F18R transcripts are indicated on the right.
FIG. 2
FIG. 2
Temporal synthesis of the A30L protein. BS-C-1 cells were mock infected for 8 h (U) or infected with VV at a multiplicity of 10 in the absence or presence of cytosine arabinoside (AraC) and harvested between 0 and 24 h postinfection (hpi). Proteins from total-cell extracts were resolved by electrophoresis on a 10 to 20% gradient polyacrylamide gel in SDS-Tricine buffer and analyzed by Western blotting using antiserum directed to the C-terminal 11 amino acids of the A30L protein. Proteins were detected by chemiluminescence. The positions of migration and molecular masses of marker proteins are indicated on the left.
FIG. 3
FIG. 3
Association of the A30L protein with purified virions. (A) Purified VV particles were sedimented on a sucrose gradient. Fractions were collected, and the proteins were resolved by electrophoresis on an SDS–4 to 20% gradient polyacrylamide gel. Proteins were visualized by silver staining. The positions of migration and molecular masses of marker proteins are indicated on the left. (B) Proteins from the sucrose gradient fractions analyzed in panel A were separated by electrophoresis on a 10 to 20% gradient polyacrylamide gel in SDS-Tricine buffer, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal A30L peptide antibody. The bands detected by chemiluminescence are shown. (C) Sucrose-gradient purified VV (108 PFU) was incubated in Tris buffer containing 1% NP-40 with or without 50 mM DTT. After centrifugation, the soluble (S) and insoluble (P) fractions were analyzed by SDS-PAGE and Western blotting using the rabbit polyclonal A30L peptide antiserum, A14L rabbit polyclonal antibody, or A10L (P4a) rabbit polyclonal antibody as indicated.
FIG. 4
FIG. 4
Construction of an A30L-inducible rVV. (A) Schematic diagram representing the genome of vA30Li. The J2R (thymidine kinase [TK]), A30L, and A56R (HA) loci are depicted. Insertions into these loci are shown below the line. Additional abbreviations: P11, a VV late promoter; P7.5, a VV early-late promoter; lacO, E. coli lac operator; lacI, E. coli lac repressor gene; T7 pol, bacteriophage T7 RNA polymerase gene; PT7, bacteriophage T7 promoter; EMC, encephalomyocarditis virus cap-independent translation enhancer element; gus, E. coli β-glucuronidase gene; gpt, E. coli guanine phosphoribosyltransferase gene. (B) Effect of IPTG on virus plaque formation. BS-C-1 cell monolayers were infected with vT7LacOI, vA30L/A30Li, or vA30Li in the presence or absence of 50 μM IPTG as indicated. Cells were stained with crystal violet at 48 h after infection.
FIG. 5
FIG. 5
Effect of IPTG on yields of vA30Li. (A) BS-C-1 cells were infected with vT7LacOI (⧫), vA30L/A30Li (▵), or vA30Li (●) at a multiplicity of 5 and incubated in the presence of 0 to 200 μM IPTG for 24 h. All virus titers were determined by plaque assay in the presence of 50 μM IPTG. (B) BS-C-1 cells were infected with vT7LacOI (⧫), vA30L/A30Li (▴), or vA30Li (●) in the absence (open symbols) or presence (filled symbols) of 50 μM IPTG. Cells were harvested at the indicated times after infection, and the total virus titer of each sample was determined as for panel A.
FIG. 6
FIG. 6
Effect of IPTG on the synthesis of the A30L protein. BS-C-1 cells were mock infected (U) or infected with vA30Li at a multiplicity of infection (MOI) of 1 or 10 in the presence of 0 to 100 μM IPTG. At 24 h after infection, the cells were harvested and the proteins were analyzed by electrophoresis on a 10 to 20% gradient polyacrylamide gel using SDS-Tricine buffer. The proteins were then transferred to a nitrocellulose membrane and incubated with the rabbit polyclonal A30L peptide antibody. The bands detected by chemiluminescence are shown. The arrow points to the A30L protein.
FIG. 7
FIG. 7
Synthesis and processing of viral proteins. (A) Pulse-labeling of viral proteins. BS-C-1 cells were infected with vT7LacOI or vA30Li at a multiplicity of 10 in the presence (+) or absence (−) of 50 μM IPTG as indicated. Cells were labeled with a mixture of [35S]methionine and [35S]cysteine for 30-min periods starting at 3, 6, 9, 12, or 24 h after infection or after mock infection (U). Immediately after labeling, the cells were washed and lysed, and the labeled proteins were denatured with SDS and mercaptoethanol and analyzed by electrophoresis on a 4 to 20% gradient polyacrylamide gel. An autoradiograph is shown. Numbers on the left correspond to molecular masses of the marker proteins. The positions of migration of proteins that are over- or underexpressed in cells infected with vA30Li in the absence of IPTG are indicated by an asterisk or a dash, respectively. (B) Proteolytic processing of viral late proteins. BS-C-1 cells were infected either with vT7LacOI in the presence (+) or absence (−) of 100 μg of rifampin (RIF) per ml or with vA30Li in the presence (+) or absence (−) of 50 μg of IPTG per ml. At 6 h after infection, the cells were pulse-labeled with a mixture of [35S]methionine and [35S]cysteine for 30 min. Cells were either harvested immediately (pulse) or incubated with excess unlabeled methionine for an additional 12 h (chase). The proteins were denatured with SDS and mercaptoethanol and analyzed by electrophoresis on a 4 to 20% gradient polyacrylamide gel and autoradiography. The positions of migration of the major core precursor protein (P4a and P4b) and their mature, processed forms (4a and 4b) are shown on the right.
FIG. 8
FIG. 8
Electron microscopy of cells infected with vA30Li in the presence or absence of IPTG. BS-C-1 cells were infected with vA30Li at a multiplicity of 10 in the presence (B) or absence (A, C, and D) of 50 μM IPTG. At 24 h after infection, the cells were fixed and prepared for transmission electron microscopy. The arrow in panel A points to a large dense granular mass that forms in the absence of IPTG. Abbreviations: C, crescents; nu, nucleoid within an IV.
FIG. 9
FIG. 9
Localization of the A30L protein by immunoelectron microscopy. BS-C-1 cells were infected with vA30LiHA at a multiplicity of 10 in the presence of 100 μg of IPTG, per ml. After 22 h, the cells were fixed in paraformaldehyde, cryosectioned, and incubated with MAb MHA.11 followed by rabbit anti-mouse IgG and protein A-conjugated to colloidal gold. Fields with numerous IV and IEV are shown in panels A and B, respectively.
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