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. 2005 Apr;79(8):4952-64.
doi: 10.1128/JVI.79.8.4952-4964.2005.

Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus

Jill T Bechtel  1 , Richard C Winant, Don Ganem
Affiliations

Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus

Jill T Bechtel et al. J Virol. 2005 Apr.

Abstract

Infection of cultured cells with Kaposi's sarcoma associated herpesvirus (KSHV) typically establishes a latent infection, in which only a few viral genes are expressed. Recently, it has been reported that a subset of lytic genes are transiently expressed very early after viral entry but that this burst of abortive lytic gene expression is terminated with the supervention of latency (H. H. Krishnan, P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B. Chandran, J. Virol. 78:3601-3620, 2004). To identify molecules imported into cells by KSHV that might influence this gene expression program, we have examined the protein composition of the KSHV particle. Immunoblotting of virus particles demonstrated that RTA, the lytic switch protein, and RAP, a viral protein that is a transcriptional and cell cycle modulator, were both incorporated into virus particles. In a second approach, polypeptides isolated from purified virions were identified by mass-spectrometric analysis of their constituent tryptic peptides. With this approach we were able to identify 18 major virion proteins, including structural, regulatory, and signaling proteins of both viral and cellular origin.

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Figures

FIG. 1.
FIG. 1.
Gradient purification of KSHV virions. Concentrated KSHV virus stocks were separated on a 20 to 35% Histodenz gradient. Fractions were collected from the bottom (fraction 1) and analyzed for viral DNA and glycoprotein content as described in Materials and Methods. (A) Plot of phosphorimager quantitation of KSHV viral DNA isolated from each fraction, spotted onto nylon membranes, and probed for the PAN locus. (B) Immunoblots of proteins isolated from each fraction, separated on 10% SDS-PAGE gels, transferred to PVDF, and probed for K8.1.
FIG. 2.
FIG. 2.
Immunoblots for viral components. Induced BCBL-1 cells and mock-treated (V), trypsin-treated (VT), and trypsin- and Triton X-100-treated (VTT) gradient-purified virions were separated on 10% SDS-PAGE gels, transferred to PVDF, and then probed for various KSHV proteins.
FIG. 3.
FIG. 3.
Coomassie blue staining of treated virions. Mock-treated (V), trypsin-treated (VT), and trypsin- and Triton X-100-treated (VTT) gradient-purified virions were separated on a SDS-7.5% PAGE gel and then stained with Coomassie blue. The destained gel is shown. Each lane represents material from ∼5 liters of induced BCBL-1 cells. Protein identifications to the right of the gel are the identifications resulting from mass spectrometric analysis of the designated bands. Bands designated as fragments represent the tryptic fragments of the protein.
FIG. 4.
FIG. 4.
Immunoblots of cellular virion components. Induced BCBL-1 cells, mock-treated virions (V), trypsin-treated virions (VT), and trypsin- and Triton X-100-treated virions (VTT) were separated on 10% SDS-PAGE gels, transferred to PVDF, and then probed with antibodies for the indicated cellular proteins.
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