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. 2008 Dec 15:5:148.
doi: 10.1186/1743-422X-5-148.

Functional characterization of the vaccinia virus I5 protein

Bethany Unger  1 , R Jeremy Nichols, Eleni S Stanitsa, Paula Traktman
Affiliations

Functional characterization of the vaccinia virus I5 protein

Bethany Unger et al. Virol J. .

Abstract

The I5L gene is one of approximately 90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5), we show here that the I5 protein is expressed as a post-replicative gene and that the approximately 9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vDeltaindI5V5), or one in which the I5 locus has been deleted (vDeltaI5), we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression.

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Figures

Figure 1
Figure 1
Tools for characterization and analysis of the I5 protein. (A) A Kyte-Doolittle hydrophilicity plot of the I5 protein was generated using the Protean module of the Lasergene software (DNASTAR, Inc.). (B) An alignment of the I5 homologs encoded by several chordopoxviruses is shown; sequences were obtained from and aligned using the Megalign module of the Lasergene software. Vaccinia virus (VV, GenBank ID 29692238), camelpox virus (CMLV, GenBank ID 18640364), ectromelia virus (EV, GenBank ID 22164721), variola virus (VAR, GenBank ID 439035), monkeypox virus (MPV, GenBank ID 179750543), Yaba-like disease virus (YLDV, GenBank ID 12085087), myxoma virus (MYX, GenBank ID 18426922), molluscum contagiosum virus (MCV, GenBank ID 1492060), Shope fibroma virus (SFV, GenBank ID 18448493), lumpy skin disease virus (LSDV, GenBank ID 151505037), sheeppox virus (ShPV, GenBank ID 21492557), and swinepox virus (SPV, GenBank ID 18640187). (C) The key genomic features of three recombinant viruses used in this study are shown. In the vI5V5 virus, the endogenous locus has been modified such that the I5 protein is fused to a C-terminal V5 epitope tag. In the inducible vΔindI5V5 recombinant, the endogenous I5 ORF has been replaced by a NEO cassette, and the tetR gene and an inducible copy of the I5V5 ORF has been inserted into the non-essential thymidine kinase (TK) locus of the genome. The inducible I5V5 ORF is under the regulation of the I5 promoter and the TET operator. The vΔI5 virus was generated by replacing the I5V5 locus of vI5V5 with the NEO cassette; this virus is deleted for the I5 locus. The primers and strategy used for the generation of these viruses are shown in Table 1.
Figure 2
Figure 2
Characterization of the endogenous I5V5 protein. (A). I5 is expressed as a late protein. Cells were left uninfected (lane 1) or infected (MOI 5) with wt virus (lane 2) or with vI5V5 in the absence (lane 3) or presence (lane 4) of AraC (cytosine arabinoside, 20 μM). Cells were harvested at 8 hpi and lysates were probed with either an anti-G7 or anti-V5 antibody. The molecular masses (in kDa) of protein standards are shown at the left. (B) I5 is not phosphorylated in vivo. BSC40 cells were infected with wt virus (lanes 1,2,5,6) or vI5V5 (lanes 3,4,7,8) (MOI 5) in the presence of either 35S-met (lanes 1–4) or 32PPi (lanes 5–8) and then harvested for immunoprecipitation analysis. Immunoprecipitation was performed with antisera specific for the F18 protein (odd numbered lanes) or the V5 epitope (even numbered lanes); immune complexes were resolved electrophoretically and visualized by audioradiography. The molecular masses (in kDa) of protein standards are shown at the right. (C) I5 shows a punctate distribution throughout the cytoplasm. BSC40 cells were infected with wt virus or vI5V5 for 8 hpi; fixed cells were stained with the anti-V5 antibody and a secondary antibody conjugated to Alexafluor 594 and DAPI. (D) I5 localizes to crescents, immature and mature virions. Cells were infected with V5I5 (MOI 2) and harvested at 17 hpi for post-embedding immunoelectron microscopy. Sections were incubated with anti-V5 antibody and secondary antibodies conjugated to 5 nm (panels B, C, E) or 10 nm (panels A, D, F) gold particles.
Figure 3
Figure 3
Analysis of the I5 protein found within mature virions. (A) I5 is encapsidated within mature virions. Increasing concentrations of purified I5V5 virions (0.5, 0.8, 2 μg) were subjected to immunoblot analysis and probed with the anti-V5 antibody. The 9 kDa I5V5 protein was readily visualized; the molecular masses (in kDa) of protein standards are shown at the right. (B). I5 is found within the virion membrane. wt and I5V5 virions purified by sedimentation on 25–40% sucrose gradients were treated with NP40 or NP40 and DTT (5). The soluble (S) and particulate (P) fractions, representing the membrane and core components, respectively, were resolved by sedimentation and analyzed by immunoblot analysis with anti-V5 and antibodies against known membrane (A17) and core (F18) proteins. (C) The V5 epitope is accessible on the surface of intact I5V5 virions. Purified vI5V5 virions (or a control virus encoding a wt I5 protein lacking the V5 epitope) were applied to grids and probed with either a control antibody (anti-A17) or the anti-V5 antibody and a secondary antibody conjugated to 10 nm gold particles. (D). Proteolytic treatment of vI5V5 virions. I5V5 virions (6 μg) were subjected to treatment with chymotrypsin (Chymo) or trypsin (Tryp) for 30 min or with proteinase K (ProtK) for 10 or 30 min. After sedimentation at 14,000 × g, 5 min, the soluble (S) and pellet (P) fractions were resolved and analyzed by immunoblot analysis using anti-D8 (top panel) or anti-V5 (lower panel) antibodies. The molecular masses (in kDa) of protein standards are shown at the right.
Figure 4
Figure 4
Repression or deletion of the I5 locus does not have a deleterious effect on virus replication in tissue culture. (A) The vΔindI5V5 virus allows tight repression of the I5 protein. BSC40 cells were infected (MOI 2) with vΔindI5V5 in the presence (lanes 1,3) or absence (lanes 2,4) of TET. Cells were harvested at 17 hpi and lysates were subjected to immunoblot analysis with the anti-V5 serum (lower panel) or antibodies specific for the intermediate and late viral proteins I3 and L4, respectively (top panel). The molecular masses (in kDa) of protein standards are shown at the right. (B) Repression of I5 does not affect the viral yield produced in a single infectious cycle in BSC40 cells or primary human fibroblasts. BSC40 cells or primary human fibroblasts were infected with vΔindI5V5 (MOI 3) in the presence or absence of TET and harvested at 24 hpi. Viral yield (pfu/ml) was determined by titration on BSC40 cells; experiments were preformed in duplicate and titrated in duplicate. Error bars represent standard deviation. (C) The vΔI5 virus is deleted for the I5 locus. Cells were infected with the parental vI5V5 virus (lanes 2,4) or with two isolates of the vΔI5 virus (lanes 1,3) at an MOI 2 and harvested at 18 hpi. Lysates were examined by immunoblot analysis with the anti-V5 antibody. The molecular masses (in kDa) of protein standards are shown at the right. (D) Deletion of I5 does not affect the viral yield produced in a single infectious cycle in BSC40 cells or primary human fibroblasts. BSC40 cells or human diploid fibroblasts were infected with wt virus, vI5V5 or vΔI5 (MOI 3) for 24 h, and the viral yield was determined as described for panel B. (E) Repression or deletion of I5 does not affect viral plaque size. BSC40 cells were infected with 50–75 PFU of vI5V5, vΔindI5V5 + TET, vΔindI5V5 – TET, or vΔI5; plates were fixed and stained with crystal violet at 48 hpi.
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