Identification of proteins associated with murine gammaherpesvirus 68 virions

Abstract

Murine gammaherpesvirus 68 (MHV68 [also known as gammaHV-68]) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.

FIG. 1.

Purification of extracellular MHV68. (A) Nucleic acids extracted from MHV68 sucrose gradient fractions. Extracellular virus was purified by 5 to 55% sucrose density gradient ultracentrifugation. Numbers shown on top of the panel are fractions collected from top (1) to bottom (13) of the gradient. Nucleic acids were extracted and electrophoretically separated in a 0.75% agarose-Tris-acetate-EDTA gel. (B) Southern blot analysis of extracellular virus. DNA shown in panel A was transferred to a positively charged nylon membrane and probed with random-primed [α³²P]dCTP-labeled virus-specific probe (a 760-bp PCR product of ORF67). + is intact viral genomic DNA. (C) Infectivity of sucrose gradient-purified virus. BHK cell monolayers infected with fractions 5, 7, or 9 were incubated in methylcellulose overlay medium at 37°C in 5% CO₂ for 5 days, and numbers of PFU were calculated. Plaque assays were performed twice. (D) Electron cryomicrograph of MHV68 virions and enveloped capsid particles. MHV68 particles from fraction 9 were embedded in vitreous ice and recorded at 100 kV on a JEOL JEM1200 electron cryomicroscope at magnification ×30,000 at a dosage of 6 electrons/angstrom². A representative image is shown, with putative virions (with DNA) indicated by an arrow (↓) and noninfectious enveloped particles (no DNA) indicated by an arrowhead (>). Bar, 100 nm.

FIG. 2.

Fractions 7 and 9 contain virion antigens. (A) Western blot using polyclonal antiserum raised in rabbits against bacterially expressed and purified viral capsid proteins (ORF26, middle panel, and ORF65, lower panel) and envelope protein (gp150, upper panel). (B) Proteins from fraction 9 are separated on SDS-8% (left) or 15% (right) PAGE and stained with SYPRO-Ruby (Molecular Probes). Bands were excised, digested in sequence-grade modified trypsin, and analyzed by liquid chromatography with tandem mass spectrometry. Proteins matching the MHV68 proteome (▵) correspond to Table 2 from high to low apparent molecular weight (mw); cellular protein matches and unidentified bands are not marked.

FIG. 3.

ORF45 is a novel virion-associated protein partly resistant to detergent (Detergt.) treatment. Virions (lane V, approximately 2,000 PFU, fraction 9) were incubated with Triton X-100 (2%) and SDS (0.1%) for 30 min at 37°C and pelleted in a tabletop centrifuge at 23°C, 20,000 × g for 25 min. The supernatant (S) and pellet (P) were removed, denatured in Laemmli buffer, and separated on an SDS-15% polyacrylamide gel. Western blots were incubated with polyclonal antisera to recombinant viral proteins: glycoprotein 150 (upper panel), ORF45 (middle panel), and ORF26 (lower panel). mw, molecular weight.