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. 2002 Nov;76(22):11216-25.
doi: 10.1128/jvi.76.22.11216-11225.2002.

Identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions

Terry A McKelvey  1 , Stanley C Andrews, Sara E Miller, Caroline A Ray, David J Pickup
Affiliations

Identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions

Terry A McKelvey et al. J Virol. 2002 Nov.

Abstract

The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.

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Figures

FIG. 1.
FIG. 1.
The p4c gene is intact in the genome of VV-WR but disrupted in the genome of CPV-BR. The rectangles correspond to the open reading frames of the ati, p4c, and 14K genes, where mutant alleles are designated ati1 and p4c1. The arrows indicate the position of the first potential initiation codon within each open reading frame. Each of these initiation codons, except for that in orf b of p4c1, is contained within a predicted late promoter element. The solid rectangle corresponds to the position of the 28 GAT repeat sequence predicted to encode a 28-aspartic-acid repeat (52). The scale is in base pairs.
FIG. 2.
FIG. 2.
The P4c protein is present in VV-WR particles but absent from particles of CPV-BR. Proteins present in virus particles (16 μg per sample), purified as described previously(36), were characterized as follows. (A) Virus particles were solubilized by boiling them for 3 min in a buffer containing 0.1 M sodium phosphate (pH 7.2), 6 M urea, 1 mM EDTA, 2% SDS, 5% β-mercaptoethanol, and 10% glycerol. The solubilized proteins were resolved by electrophoresis in a 10% polyacrylamide gel containing 0.1 M sodium phosphate, 6 M urea, 1 mM EDTA, and 0.1% SDS (61) and visualized with Coomassie brilliant blue R-250 stain. (B) Virus particles were solubilized by boiling them for 3 min in a buffer containing 50 mM Tris (pH 7.0), 1% SDS, 30 mM dithiothreitol, 5 mM EDTA, and 10% glycerol. The solubilized proteins were resolved by electrophoresis in a 10% polyacrylamide gel using a discontinuous buffer system as described previously (43) and visualized with Coomassie brilliant blue R-250 stain. Lanes 1, proteins of CPV-BR; lanes 2, proteins of VV-WR; lanes 3, protein markers (in kilodaltons).
FIG. 3.
FIG. 3.
Insertional inactivation of the p4c gene leads to loss of intact P4c protein in the purified virus. Proteins present in virus particles (16 μg per sample), purified as described previously(36), were characterized as described in the legend to Fig. 2. (A) Solubilized proteins were resolved by electrophoresis in an 8% polyacrylamide gel containing a continuous phosphate buffer system (61). (B) Solubilized proteins were resolved by electrophoresis in a 12.5% polyacrylamide gel using a discontinuous buffer system as described previously (43). Lanes 1, protein markers (sizes in kilodaltons); lanes 2, proteins of VV-WR; lanes 3, proteins of vaccinia virus A504; lanes 4, proteins of CPV-BR. The arrow indicates the position of an ∼34-kDa protein present only in vaccinia virus A504 (panel B, lane 3).
FIG. 4.
FIG. 4.
Expression of the P4c protein in cells infected with recombinant vaccinia and cowpox viruses. Human 143B cells were infected and metabolically labeled with [35S]methionine for 30 min 18 h after infection, as described in Materials and Methods. The solubilized labeled proteins were resolved by electrophoresis in a 12.5% polyacrylamide gel using a discontinuous buffer system as described previously (43) and visualized by autoradiography of the dried gel. The lanes contain proteins from cells infected with VV-WR (lane 1), vaccinia virus A504 (lane 2), vaccinia virus A507 (lane 3), cowpox virus A505 (lane 4), and CPV-BR (lane 5). Lane 6 contains protein markers (in kilodaltons).
FIG. 5.
FIG. 5.
VV-WR expressing the p4c gene can provide a factor needed to direct the inclusion of IMV within ATIs. Human 143B cells were infected with 10 PFU of one strain of poxvirus/cell (A and C) or with 5 PFU each of two strains of poxvirus/cell (B and D). Eighteen hours after the cells were infected, they were fixed, stained, sectioned, and examined by electron microscopy to identify the phenotype of the ATIs present in the infected cells. Shown are sections of cells infected with VV-WR (p4c ati1) (A); VV-WR and CPV-BR (p4c1 ati), where the large mass is a V+ ATI (B); vaccinia virus Copenhagen [Φ(p4c′-Δati)] (C); and vaccinia virus Copenhagen and CPV-BR, where the large mass is a V− ATI (D). Bars, 1,000 nm.
FIG. 6.
FIG. 6.
The p4c gene is required for the formation of V+ ATIs in cells coinfected with VV-WR and CPV-BR. Human 143B cells were infected with either 10 PFU of one strain of poxvirus/cell (A and C) or 5 PFU each of two strains of poxvirus/cell (B and D). Eighteen hours after the cells were infected, they were fixed, stained, sectioned, and examined by electron microscopy. Shown are sections of cells infected with vaccinia virus A504 (p4c::gpt ati1) containing a p4c gene disrupted by the insertion of a selectable marker gene, the gpt gene (A); vaccinia virus A504 and CPV-BR (p4c1 ati), where the large masses are V− ATIs (B); vaccinia virus A507 (p4c ati1), which is a revertant of vaccinia virus A504 containing an intact p4c gene (C); and vaccinia virus A507 and CPV-BR, where the large masses are V+ ATIs (D). Bars, 1,000 nm.
FIG. 7.
FIG. 7.
Insertion of the vaccinia virus p4c gene into the genome of CPV-BR is sufficient to convert the phenotype of the ATIs from V− to V+. Human 143B cells were infected with 10 PFU of cowpox virus/cell. Eighteen hours after infection, the cells were fixed, stained, sectioned, and examined by electron microscopy. Shown are sections of cells infected with CPV-BR (p4c1 ati), which produces V− ATIs (A), and cowpox virus A505 (p4c1 p4c ati), which is a recombinant cowpox virus containing the VV-WR p4c gene under the control of its own promoter (B). ATIs in cells infected with cowpox virus A505 have a V+ phenotype. Bars, 1,000 nm.
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