Identification of Proteins Associated with Murine Cytomegalovirus Virions

Abstract

Proteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by complete denaturation and subjected to either separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel digestion or treated directly by in-solution tryptic digestion. Peptides were separated by nanoflow liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against a database of MCMV open reading frames (ORFs) predicted to be protein coding by an MCMV-specific version of the gene prediction algorithm GeneMarkS. We identified 38 proteins from the capsid, tegument, glycoprotein, replication, and immunomodulatory protein families, as well as 20 genes of unknown function. Observed irregularities in coding potential suggested possible sequence errors in the 3-proximal ends of m20 and M31. These errors were experimentally confirmed by sequencing analysis. The MS data further indicated the presence of peptides derived from the unannotated ORFs ORF c225441-226898 (m166.5) and ORF 105932-106072 . Immunoblot experiments confirmed expression of m166.5 during viral infection.Murine cytomegalovirus (MCMV), a member of the betaherpesvirus family, shares 45.2% sequence identity with human CMV (HCMV) and is currently the most commonly used animal model for the study of CMV-induced disease. The MCMV genome has a size of 230 kbp and was originally estimated to encode 170 proteins (45). The MCMV virion consists of double-stranded viral DNA surrounded by an icosahedral capsid, a complex proteinaceous tegument, and a lipid membrane (34). Although the composition of HCMV particles has been addressed (3, 10), the protein composition of the MCMV virion has not been examined in detail.The majority of MCMV genes have been annotated based on their homologues in HCMV (45) and, consequently, many open reading frames (ORFs), including genes encoding some structural proteins, lack experimental confirmation. Analysis of regions unique to MCMV has been even more limited. There is, therefore, a need for characterization of the composition of MCMV particles, as well as the confirmation of putative structural and functional homologues of HCMV gene products.The traditional method of analyzing the protein composition of a virus preparation consists of denaturation in sodium dodecyl sulfate (SDS), separation of the constituent proteins by electrophoresis, and identification either by immunological methods or by sequencing of visible bands via Edman degradation or mass spectrometry (MS). This approach has been used to analyze other herpesvirus family members, such as HCMV (3), murine gammaherpesvirus 68 (9), Epstein-Barr virus (18), and Kaposi's sarcoma-associated herpesvirus (36). Direct digestion of viral particles and "shotgun" sequencing by liquid chromatography-tandem MS (LC-MS/MS) was first explored with the much simpler adenovirus type 5 proteome (13). Although adenovirus p...

Actin and Rho GTPases in herpesvirus biology

“…Recent work has shown that host-derived actin is incorporated into the PRV virion and becomes an integral part of the outer tegument layer [42,43]. Virion-associated actin has been reported in other herpesviruses [44][45][46][47][48][49] and other enveloped viruses, including paramyxovirus, retrovirus and rhabdovirus [50][51][52][53][54]. But why do herpesviruses contain F-actin in the virion?…”

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

“…In recent years, mass spectrometry (MS)-based analysis has been used to identify the composition of different herpesviruses (2,3,7,13,25,27,28,31,41,53,55,58). Within rhadinoviruses, KSHV (3,58), rhesus monkey rhadinovirus (RRV) (41), and murid herpesvirus 4 (MuHV-4) (7) extracellular virions have been analyzed using this approach.…”

Annexins in Translational Research: HIDDEN Treasures to Be Found

“…In addition to IAV, AnxA1 is found associated with several other viruses [114][115][116][117][118][119][120][121][122]. Interestingly, no indications so far suggest that the viruses rely on its immune-modulating capacity to facilitate virus entry, although the AnxA1 receptor FPR2 is involved in IAV replication [123].…”