Vaccinia virus morphogenesis: a13 phosphoprotein is required for assembly of mature virions

Abstract

The 70-amino-acid A13L protein is a component of the vaccinia virus membrane. We demonstrate here that the protein is expressed at late times of infection, undergoes phosphorylation at a serine residue(s), and becomes encapsidated in a monomeric form. Phosphorylation is dependent on Ser40, which lies within the proline-rich motif SPPP. Because phosphorylation of the A13 protein is only minimally affected by disruption of the viral F10 kinase or H1 phosphatase, a cellular kinase is likely to be involved. We generated an inducible recombinant in which A13 protein expression is dependent upon the inclusion of tetracycline in the culture medium. Repression of the A13L protein spares the biochemical progression of the viral life cycle but arrests virion morphogenesis. Virion assembly progresses through the formation of immature virions (IVs); however, these virions do not acquire nucleoids, and DNA crystalloids accumulate in the cytoplasm. Further development into intracellular mature virions is blocked, causing a 1,000-fold decrease in the infectious virus yield relative to that obtained in the presence of the inducer. We also determined that the temperature-sensitive phenotype of the viral mutant Cts40 is due to a nucleotide transition within the A13L gene that causes a Thr(48)-->Ile substitution. This substitution disrupts the function of the A13 protein but does not cause thermolability of the protein; at the nonpermissive temperature, virion morphogenesis arrests at the stage of IV formation. The A13L protein, therefore, is part of a newly recognized group of membrane proteins that are dispensable for the early biogenesis of the virion membrane but are essential for virion maturation.

FIG. 1.

Structural organization and viral conservation of A13 protein. (A) Structural features of A13 protein. A hydrophobicity plot is shown above the A13 sequence; the N terminus (aa 1 to 20) contains a predicted transmembrane domain. The region marked with the blue line was used to generate the anti-A13 serum. Residues of interest are shown in color as follows: red, Lys; purple, Glu; blue, numerous Asn residues; green, numerous, clustered Pro residues. The eight Ser residues are marked with circles; the red diamond at Ser40 marks the site of A13 phosphorylation (Fig. 8); the red arrowhead marks the Ile residue that is altered in Cts40 (Table 2 and Fig. 9). (B) Sequence alignment of poxviral A13 homologs. A sequence alignment of the A13 orthologs from vaccinia virus (VV; GenBank ID 29692238), cowpox virus (CPV; GenBank ID 20178507), camelpox virus (CMPV; GenBank ID 18640364), monkeypox virus (MPX; GenBank ID 17975037), variola virus (VAR; GenBank ID 439035), ectromelia virus (EV; GenBank ID 22164721), lumpy skin disease virus (LSDV; GenBank ID 15150543), sheeppox virus (ShPV; GenBank ID 21492557), swinepox virus (SPV; GenBank ID 18640187), Yaba-like disease virus (YLDV; GenBank ID 12085087), and molluscum contagiosum virus (MCV; GenBank ID 1492060) (sequences obtained from www.poxvirus.org by using the POCs program) is shown. Residues identical to A13 are boxed and shaded in yellow.

FIG. 2.

Expression, encapsidation, and posttranslational modification of A13. (A) Temporal expression of A13. BSC40 cells were left uninfected (lane 1) or infected with wt virus (MOI = 15) in the presence or absence of AraC and were metabolically labeled with [³⁵S]methionine for 30 min prior to being harvested. Protein lysates were prepared and subjected to immunoprecipitation with anti-A13 serum. The lanes correspond to immunoprecipitates retrieved from the following extracts: mock-infected cells (lane 1); cells infected with wt virus and harvested at 1.5, 2.75, 4, 5.25, and 6.5 hpi (lanes 2 to 6); and cells infected with wt virus in the presence of AraC and harvested at 4 hpi (lane 7). The arrow indicates A13. The electrophoretic migration of 18.4- and 14.3-kDa protein standards is shown at the left. (B) Encapsidation of A13 protein. Purified wt virions were resuspended in protein sample buffer in the presence (+) or absence (−) of β-mercaptoethanol (β-ME), boiled, resolved by SDS-17% PAGE, and subjected to immunoblot analysis with anti-A13 serum. The position of the monomeric form of A13 is indicated with an arrow. Protein standards of 18.4, 14.3, and 6.0 kDa are indicated on the left. (C) Phosphorylation of A13 protein in vivo. Cells were infected with wt virus at 37°C (lanes 1), 31.5°C (wt³²; lanes 3), or 39.7°C (wt⁴⁰; lanes 4), with vindH1 in the absence of IPTG (H1⁻; lanes 2), or with ts28 at 31.5°C (tsF10³²; lanes 5) or 39.7°C (tsF10⁴⁰; lanes 6). All cultures were infected at an MOI of 2 and were radiolabeled with ³²PPi from 3 to 18 hpi. The lysates were subjected to immunoprecipitation analysis with anti-A13 serum (left panel), anti-A17 serum (center panel), or anti-I3 serum (right panel). The positions of A13, A17, and I3 are indicated by arrows; coprecipitated ³²P-A14 (center panel, lane 2) is denoted by a diamond. Immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography. Lane M contains protein standards, with their molecular masses shown at the right, in kilodaltons.

FIG. 3.

Phenotypic analysis of vindA13. (A) Plaque assay. Confluent BSC40 cells were infected with two different dilutions of vindA13 (∼500 and 50 PFU/dish) and maintained in the presence (+) or absence (−) of TET (1 μg/ml). At 48 hpi, the medium was removed and the cells were stained with crystal violet. (B) Quantitation of 24-h viral yield. Cells were infected with vindA13 (MOI = 2) in the presence (+) or absence (−) of TET and harvested at 24 hpi. The yield of total cell-associated virus was determined by titration on BSC40 cells in the presence of TET. (C) Quantitation of A13 accumulation. Cells were infected with vindA13 (MOI = 2) in the presence (+ [lower panel]) or absence (− [upper panel]) of TET (1 μg/ml) and harvested at 24 hpi. Serial dilutions of total cell extracts were resolved by SDS-17% PAGE and subjected to immunoblot analysis with anti-A13 serum. The position of A13 is indicated with an arrow. (D) S1 nuclease analysis of A13-specific and read-through mRNA transcripts. A schematic diagram of the 371-nt radiolabeled probe used for the S1 nuclease analysis and of the transcripts expected for vtetR and vindA13 infections is shown at the top. The position of the 5′ radiolabel at the BamHI site is marked with a star; the probe contains the TET operator (triangle) inserted between the transcriptional (TAAATA) and translational (ATG) start sites of the A13 gene. The wavy lines depict the 5′ regions of the mRNAs that are predicted to be expressed during vindA13 (top) or vtetR (bottom) infections. The probe fragment protected by hybridization with the vindA13 transcript should be slightly larger than that protected by the vtetR transcript due to the presence of the 19-bp operator sequence (285 versus 263 nt) in the vindA13 allele. The autoradiograph below shows the results of the S1 nuclease protection assay. RNA was prepared from cells infected for 8 h with vtetR or vindA13 (MOI = 10) in the presence (+) or absence (−) of TET (1 μg/ml). Reactions were performed, electrophoretically resolved, and visualized by autoradiography as described in Materials and Methods. Lanes 1 and 2, control reactions in which the probe was incubated in the absence of RNA, without or with subsequent S1 nuclease digestion, respectively; lanes 3 and 4, results obtained with RNA prepared from cells infected with vindA13 in the presence or absence of TET, respectively. Protection of the full-length probe is seen with both RNA samples, but only the sample from the +TET infection contains transcripts that generate a protected fragment of 285 nt (open triangle, compare lanes 3 and 4). Lanes 5 and 6 depict the results obtained with RNA prepared from cells infected with vtetR in the presence or absence of TET, respectively. In both cases, protection of the full-length probe and the 263-nt A13-specific fragment (filled triangle) is seen.

FIG. 4.

Phenotypic characterization of vΔindA13. (A) Plaque assay. vtetR and vΔindA13 were titrated on BSC40 cells in the presence (+) or absence (−) of TET. At 48 hpi, the medium was removed and the cells were stained with crystal violet. (B) Quantitation of 24-h viral yield. Cells were infected with vtetR or vΔindA13 (MOI = 2) for 24 h in the presence (+) or absence (−) of TET; cell lysates were prepared and the virus was titrated onto BSC40 cells in the presence of TET. (C) Quantitation of A13 accumulation. Cells were infected with vtetR (left) or vΔindA13 (right) (MOI = 2) in the presence (+) or absence (−) of TET (1 μg/ml) and harvested at 24 hpi. Serial dilutions of total cell extracts were resolved by SDS-17% PAGE and subjected to immunoblot analysis with anti-A13 serum. The position of A13 is indicated with an arrow; protein standards of 14.8 and 6.0 kDa are shown at the left.

FIG. 5.

Impact of A13 repression on temporal profile of protein synthesis, genome resolution, and proteolytic processing. (A) Temporal profile of viral protein synthesis. BSC40 cells were infected with vΔindA13 (with or without TET) at an MOI of 5 and were metabolically labeled with [³⁵S]methionine for 45 min before being harvested at the indicated time points (2, 4, 6, and 8 hpi). Mock-infected cells were also labeled with [³⁵S]methionine for 45 min before being harvested (lane 1). Lysates were resolved by SDS-12% PAGE and visualized by autoradiography. The molecular masses of ¹⁴C-protein standards are indicated on the right, in kilodaltons. Representative intermediate proteins are indicated by ovals, late proteins are indicated by arrowheads, and cellular actin is indicated by a square. (B) Southern blot analysis of genome resolution. BSC40 cells were infected with wt virus or vΔindA13 (MOI = 2) in the presence (+) or absence of TET. As a control, cells were also infected with wt virus in the presence (+) of IBT or with vROG8 (MOI = 15) in the absence (−) of IPTG. At 18 hpi, the cells were harvested and viral genomic DNA was isolated, digested with BstEII, resolved electrophoretically, and subjected to Southern blot analysis by hybridization with a radiolabeled probe derived from the termini of the viral genome. The arrowheads point to the 1.3-kb fragment released from mature, monomeric genomes and the 2.6-kb junction fragment released from unresolved concatemers. The probe also hybridizes to the ∼5.5-kb BstEII fragment proximal to the telomeric regions; the slight variation in the size of this fragment in different plaque-purified isolates represents variability in the numbers of tandem repeats. The sizes of DNA standards are indicated in kilobase pairs to the left. (C) Proteolytic processing of core proteins. BSC40 cells were infected (MOI = 2) with wt virus in the presence (+) (lanes 7 and 8) or absence (lanes 1 and 2) of RIF or with vΔindA13 in the presence (+) (lanes 3 and 4) or absence (−) (lanes 5 and 6) of TET. At 8 hpi, the cells were metabolically labeled with [³⁵S]methionine for 45 min before being harvested immediately (pulse [P]) or refed with complete medium and incubated for an additional 15 h (chase [C]). Cell lysates were resolved by SDS-12% PAGE and visualized by autoradiography. ¹⁴C-labeled protein standards are indicated at the right, with their molecular masses shown in kilodaltons. Precursor forms of the major core proteins 4a, 4b, and L4 are indicated by filled arrowheads; mature, processed forms are indicated by open arrowheads.

FIG.6.

Electron microscopic analysis of vΔindA13 infections. BSC40 cells were infected with vΔindA13 (MOI = 2) in the presence (A and B) or absence (C to G) of TET, and at 18 hpi they were processed for conventional transmission electron microscopic analysis. During permissive infections, numerous groupings of IV (A, arrows), IVN (A, asterisks), and IMV (B) were seen. During nonpermissive infections, IVN and IMV were rare. Small virosomes (V) surrounded by normal crescents (C) as well as irregular fragments of membranes (arrowheads) were numerous (C to F). Immature virions were also highly abundant. Finally, these infections were characterized by the presence of many dense crystalloids (stars), which are thought to contain tightly packed viral genomes (G). Final magnifications: (A) ×25,000; (B) ×21,000; (C) ×15,000; (D) ×23,000; (E) ×44,000; (F) ×60,000; (G) left panel, ×35,000; top center panel, ×52,000; lower center panel, ×45,000, ×54,000, and ×50,000; right panel, ×43,000.

FIG.7.

Electron microscopy of wt and vΔindA13 (−TET) infections after release from a RIF-induced morphogenesis arrest. Cells were infected with either wt virus or vΔindA13 (−TET; MOI = 2) in the presence of RIF. At 12 hpi, the cells were either harvested immediately (0 min) or the RIF was washed out and the infection was allowed to proceed for up to 180 min postrelease. Representative images of wt infections are shown in the top five panels. After RIF arrest (0 min), there were numerous small virosomes (V) surrounded by large fragments of irregular, flaccid membranes (filled arrowheads). By 10 min after release, crescents (C) had formed; by 90 min, both crescents and immature virions (arrows) were present. At 180 min postrelease, IVN (stars) and mature virions (empty arrowheads) were abundant. Representative images of vΔindA13 infections are shown in the bottom five panels. After RIF arrest, the overall profile was similar to that shown above for the wt virus; at 0 min, there were numerous small virosomes (V) surrounded by fragments of irregular, flaccid membranes (filled arrowheads). By 10 min after release, crescents (C) had formed; by 90 min, both crescents and immature virions (arrows) were seen. At 180 min, immature virions (arrows) were numerous, but those with nucleoids were extremely rare and virtually no mature virions were seen. DNA crystalloids (stars) were frequent. Final magnifications for wt panels: 0 min, ×30,000; 10 min, ×20,000; 90 min, ×30,000; 180 min, ×30,000 (left) and ×40,000 (right). Final magnifications for vΔindA13 panels: 0 min, ×15,000; 10 min, ×31,000; 90 min, ×35,000 X (upper right), ×23,000 (lower left); 180 min, ×38,000.

FIG. 8.

Characterization of phosphorylation of epitope-tagged alleles of A13 containing Ser→Ala substitutions. Cells were infected with vΔindA13 (MOI = 2) in the absence of TET; at 3 hpi, cells were transfected in duplicate with empty vector (V, lane 1) or with plasmids encoding 3X-FLAG-tagged wt A13 (lane 2) or alleles containing Ser→Ala substitutions at the positions shown above the lanes (lanes 3 to 8). Cells were metabolically labeled with ³²PPi from 6 to 24 hpi prior to being harvested and analyzed either by immunoprecipitation (A and B) or immunoblot analysis (C) with α-FLAG serum. Proteins were visualized by autoradiography (A and B) or chemiluminescence (C).

FIG. 9.

Phenotypic analysis of cells infected with Cts40. (A) Plaque assay. Confluent BSC40 cells were infected with either wt virus or Cts40 and incubated at 31.5 or 39.7°C for 2 days before being stained with crystal violet. (B) Quantitation of 24-h viral yield. Cells were infected with wt virus or Cts40 at either 31.5 or 39.7°C for 24 h; the total yield of cell-associated virus was determined by titration on BSC40 cells at 31.5°C. (C) Accumulation of A13 and other late proteins. BSC40 cells were infected with wt virus (lanes 1 and 4), vΔindA13 (−TET) (lanes 2 and 5), or Cts40 (lanes 3 and 6) at either 31.5°C (lanes 1 to 3) or 39.7°C (lanes 4 to 6) for 18 h. Lysates were prepared and subjected to immunoblot analysis using anti-G7, anti-L4, or anti-A13 sera.