Identification by Mass Spectrometry and Immune Response Analysis of Guinea Pig Cytomegalovirus (GPCMV) Pentameric Complex Proteins GP129, 131 and 133

Abstract

Development of a vaccine against congenital infection with human cytomegalovirus (HCMV) is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC) of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV), consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

“…Both N13R10 and ⌬145 have a 4-bp deletion that frameshifts the gp129 ORF (25) and renders these viruses unable to assemble a pentameric complex (PC) composed of gH, gL, gp129, gp131, and gp133 (42)(43)(44)52). In this respect, they resemble the HCMV Towne strain variants that comprise the Towne vaccine, which also contains a mutation that disrupts expression of a PC subunit (17).…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…Yamada et al [117] first described a region of the GPCMV genome which appeared to contain homologues of HCMV PC and, more recently, Auerbach et al [118] concluded that the guinea pig genes GP129, GP131 and GP 133 are the homologues of the HCMV genes UL128, UL130 and UL131, and that the relevant recombinant proteins are immunoreactive with convalescent sera from infected animals [119]. Thus, the protein products of GPCMV GP129, 131 and 133 locus were referred to as the GP PC, and the mutations acquired during passaging in fibroblasts attenuated virus pathogenicity [120], whereas repair resulted in congenital transmission, intrauterine growth restriction and elevated pup mortality.…”

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

“…Western blot assays were conducted using standard protocols, with virus particles purified as described previously (15). Virus particles from N13R10, r129, and SG virus (passaged once in GPL cells; 10 g per lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed using antibodies specific for GPCMV proteins ( Fig.…”

“…GPCMV glycoprotein B (gB) was detected using murine monoclonal anti-gB antibodies IE3-21 (3) or 29.29. Rabbit and guinea pig antisera specific to GP129, GP131, and GP133 are described elsewhere (15). The guinea pig antiserum to GP129 was raised against a glutathione S-transferase (GST) fusion protein, including GP129 residues Asn 146 through Lys 179 of the GP129 ORF, and was therefore not predicted to react with any putative truncated forms of GP129 corresponding to the first 101 codons of the ORF that might be expressed in the context of the 4-bp deletion mutation.…”

“…Based on these comparisons to SG virus, adult guinea pigs tolerate a 5,880-fold-higher dose of N13R10 without significant illness or mortality, while a 140-foldhigher dose of N13R10 during pregnancy produces lower rates of fetal transmission and pup loss (11,12). The molecular basis for the attenuation of N13R10 is uncertain, but DNA sequence comparisons of the SG virus and N13R10 genomes identified only two differences resulting in alteration of annotated protein coding sequences: (i) a 4-bp deletion that disrupts overlapping genes GP129 and GP130 and (ii) a missense mutation in GP130 3= of the 4-bp deletion/frameshift (14,15).…”