Virion proteins of Kaposi's sarcoma-associated herpesvirus

Abstract

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.

FIG. 1.

Protein composition of gradient-purified KSHV virions. Purified virion lysate was resolved on 3 to 8% Tris-acetate (lane 2) and 12% Bis-Tris NuPAGE (lane 4) gels with molecular markers (lanes 1 and 3) and stained with colloidal Coomassie G-250. Prominent bands were excised, digested in gel with trypsin, and subjected to LC-MS/MS analysis. The resultant MS/MS spectra were run against a sequence database with the SEQUEST program. Matched KSHV or cellular proteins are indicated at the right of each gel.

FIG. 2.

Association of KSHV putative virion proteins with purified virions. Uninduced and TPA-induced BCBL-1 cell extracts (lanes 1 and 2) and gradient-purified virions (lane 3) were resolved by SDS-4 to 12% PAGE, transferred onto nitrocellulose membranes, and immunoblotted with mouse monoclonal antibody against ORF45 (A), mouse polyclonal antibody against ORF21 (TK) (B), mouse polyclonal antibody against ORF33 (C), mouse polyclonal antibody against ORF11 (D), mouse polyclonal antibody against ORF64 (E), rabbit polyclonal immunoglobulin G against gB (F), rabbit polyclonal antibody against RTA/ORF50 (G), and rabbit polyclonal antibody against LANA (H). The values on the left are molecular sizes in kilodaltons.

FIG. 3.

Effect of trypsin digestion and detergent treatment on the association of putative tegument proteins with purified virions. Purified virions were treated with trypsin either in the absence (lane 1) or in the presence (lane 2) of 1% Triton X-100 for 1 h at 37°C or left untreated (lane 3). The proteolysis reactions were terminated by addition of 0.5 mM PMSF and 1/100 volume of protease inhibitor. The samples were analyzed by Western blotting with antibodies as indicated. In addition, virions were left untreated (lane 4) or treated with 1% Triton X-100 plus 0.5% DOC for 30 min and then centrifuged at 100,000 × g for 1 h. The supernatant (Sup) (lane 5) and pelleted tegument-capsid (lane 6) were analyzed by Western blotting.

FIG. 4.

Matches of the peptides identified by MS/MS from 43- and 240-kDa protein bands, respectively, with human nonmuscle β-actin and class II myosin heavy chain type A. The peptides identified by MS/MS from each band and their respective sequences (shaded regions) are shown superimposed on the amino acid sequences of the β-actin (A) and myosin (B) proteins, respectively.

FIG. 5.

Effect of trypsin digestion and detergent treatment on association of cellular cytoskeletal proteins with purified KSHV virions. Purified virions were treated with trypsin either in the absence (lane 1) or in the presence (lane 2) of 1% Triton X-100 for 1 h at 37°C or left untreated (lane 3). The proteolysis reactions were terminated by addition of 0.5 mM PMSF and 1/100 volume of protease inhibitor. The samples were analyzed by Western blotting with antibodies against β-actin and class II myosin heavy peptide A, respectively. In addition, virions were left untreated (lane 4) or treated with 1% Triton X-100 plus 0.5% DOC for 30 min and then centrifuged at 100,000 × g for 1 h. The supernatant (Sup) (lane 5) and pelleted tegument-capsid (lane 6) were analyzed by Western blotting. The values on the left are molecular sizes in kilodaltons.