Regulation of vaccinia virus morphogenesis: phosphorylation of the A14L and A17L membrane proteins and C-terminal truncation of the A17L protein are dependent on the F10L kinase

Abstract

This study focused on three vaccinia virus-encoded proteins that participate in early steps of virion morphogenesis: the A17L and A14L membrane proteins and the F10L protein kinase. We found that (i) the A17L protein was cleaved at or near an AGX consensus motif at amino acid 185, thereby removing its acidic C terminus; (ii) the nontruncated form was associated with immature virions, but only the C-terminal truncated protein was present in mature virions; (iii) the nontruncated form of the A17L protein was phosphorylated on serine, threonine, and tyrosine residues, whereas the truncated form was unphosphorylated; (iv) nontruncated and truncated forms of the A17L protein existed in a complex with the A14L membrane protein; (v) C-terminal cleavage of the A17L protein and phosphorylation of the A17L and A14L proteins failed to occur in cells infected with a F10L kinase mutant at the nonpermissive temperature; and (vi) the F10L kinase was the only viral late protein that was necessary for phosphorylation of the A17L protein, whereas additional proteins were needed for C-terminal cleavage. We suggest that phosphorylation of the A17L and A14L proteins is mediated by the F10L kinase and is required to form the membranes associated with immature virions. Removal of phosphates and the A17L acidic C-terminal peptide occur during the transition to mature virions.

FIG. 1

C-terminal cleavage of the A17L protein. (A) The A17L ORF is shown with the sequences used to generate synthetic peptides for immunization underlined and a slash at the known AGA and predicted AGN cleavage motifs. The antibodies induced by peptides TEEQQQSFMPKD and IPTFNSLNTDDY are referred to as A17L N antibody and A17L C antibody, respectively. (B) Western blot of extract from uninfected cells (U) and cells infected with vaccinia virus (I) and probed with A17L N antibody (anti-N) and A17L C-antibody (anti-C). The masses and positions of marker proteins are indicated on the left. Close inspection reveals that the 23- to 25-kDa bands from infected cells detected with N and C antibodies are doublets. The C antibody cross-reacted with more slowly migrating bands from uninfected and infected cells. (C) Western blot of proteins from 11 μg of sucrose gradient-purified vaccinia virions probed with A17L N and C antibodies.

FIG. 2

Immunogold labeling of viral membranes with antibodies to the A17L protein. BS-C-1 cells that had been infected with vaccinia virus for 24 h were fixed in paraformaldehyde, cryosectioned, and incubated with A17L N antibody (N) or A17L C antibody (C) and then with 10-nm-diameter gold particles conjugated to protein A. Electron microscopic images are shown with a 1-μm marker. m, mature particles; i, immature particles.

FIG. 3

C-terminal truncation of the A17L protein in the presence of rifampin. Replicate wells containing BS-C-1 cells were infected with vaccinia virus and incubated at 37°C in the absence (A) or continued presence (B) of rifampin. At 6 h after infection, the cells were incubated for 30 min with [³⁵S]methionine. The cells were then washed and incubated with medium containing unlabeled amino acids. Cells were harvested after the 30-min pulse (time zero) and after 0.5, 1, 2, and 3 h of chase as indicated, lysed, and incubated with A17L C antibody, A17L N antibody, or antibody to the 4b core protein. The bound proteins were analyzed by SDS-PAGE and autoradiography. The positions and masses of marker proteins are indicated.

FIG. 4

C-terminal truncation of the A17L protein failed to occur in cells infected at the nonpermissive temperature with an F10L mutant. Replicate BS-C-1 cells were infected with ts15 at 32°C (A) or 39.5°C (B). At 6 h after infection, the cells were incubated for 30 min with [³⁵S]methionine. The cells were then washed and incubated with medium containing unlabeled amino acids. Care was taken to maintain the temperature at 32 or 39.5°C during the labeling and chase periods. Cells were harvested after the 30-min pulse (time zero) and after 0.5, 1, 2, and 3 h of chase as indicated, lysed, and incubated with A17L C antibody or A17L N antibody. The bound proteins were analyzed by SDS-PAGE and autoradiography. The positions and masses of marker proteins are indicated.

FIG. 5

Coimmunoprecipitation of A17L and A14L proteins. Pulse-chase [³⁵S]methionine-labeling experiments were carried out as described in the legend to Fig. 4 except that cells were infected with vA17LΔ5 virus in the absence (−) or presence (+) of IPTG at 37°C and the chase was continued for 4 h. Lysates were immunoprecipitated with A17L N antibody (A) or A14L C antibody (B) and analyzed by SDS-PAGE and autoradiography. In panel A, no pulse-labeled protein was detected in the absence of IPTG, and so only the chase samples in the presence of IPTG are shown. The positions and masses of marker proteins are indicated.

FIG. 6

Phosphorylation of the A17L and the A14L proteins. BS-C-1 cells were infected with vaccinia virus vA17LΔ5 at 37°C in the presence (+) or absence (−) of IPTG or with vaccinia virus ts15 virus at the permissive (32°C) or nonpermissive (39.5°C) temperature (T) and then labeled overnight with [³⁵S]methionine (A) or with ³²P_i (B). Immunoprecipitation was performed with A17L N antibody (A17L) or with A14L C antibody (A14L) as indicated. The bound proteins were analyzed by SDS-PAGE and autoradiography. U, uninfected cells. Positions and masses of marker proteins are shown on the right.

FIG. 7

Phosphorylation of the A17L protein in cells transfected with plasmids expressing the A17L and F10L genes. BS-C-1 cells were infected with vTF7-3 in the absence or presence of AraC and transfected with pVOTE.1-A17L (A17L) alone or together with.pVOTE.2-F10L (F10L). The cells were metabolically labeled with [³⁵S]methionine (A) or ³²P_i (B), and the lysates were immunoprecipitated with A17L N antibody and analyzed by SDS-PAGE and autoradiography.

FIG. 8

SDS-PAGE analysis of proteins immunoprecipitated with antibodies to phosphorylated amino acids. BS-C-1 cells were infected with vA17LΔ5 virus in the absence (−) or presence (+) of IPTG and labeled with ³²P_i. Lysates were immunoprecipitated with antibody to phosphoserine (PS), phosphotyrosine (PY), or phosphothreonine (PT) or with A17L N-antibody (A17L) and analyzed by SDS-PAGE. The positions and masses in kilodaltons of marker proteins are shown on the left.

FIG. 9

Phosphoamino acid analysis. BS-C-1 cells were infected with vaccinia virus vA17LΔ5 in the presence of IPTG and labeled overnight with ³²P_i. After lysis and immunoprecipitation with A14L antibody, A17L N antibody, or antibody to phosphotyrosine (anti-PY), the proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. The radioactively labeled bands were excised, hydrolyzed with HCl, and analyzed by two-dimensional thin-layer electrophoresis at pH 1.9 and 3.5. The standards amino acids were visualized with ninhydrin, and the plates were autoradiographed. The spots corresponding to phosphoserine (PS), phosphothreonine (PT), phosphotyrosine (PY), P_i, phosphoribose (Pr), and phosphouridine (Up) are identified. P.peptides, phosphorylated peptides.