Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo

Abstract

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.

Viral G Protein–Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases

“…Analyses of murine, rat, guinea pig, and rhesus cytomegaloviruses are also yielding an understanding of the roles of CMV GPCRs in vivo (Beisser et al, 1998;Kaptein et al, 2003;Case et al, 2008;Alcendor et al, 2009;Cardin et al, 2009;Takeda et al, 2018;Frank et al, 2019). The HCMV GPCRs UL33 and UL78 are well conserved across mammalian species, but homologs of US27 and US28 are only found in primate CMVs, with five homologs of US28 in rhesus CMV (Alcendor et al, 2009).…”

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals

“…Viral DNA samples in culture supernatants were prepared using a commercial kit (QIAamp Viral RNA kit). The quantitative PCR assays targeting the GP83 gene for quantification of GPCMV DNA and the guinea pig β-actin gene were described previously [9].…”

“…Some of the evasion mechanisms employed by CMV are also reported for GPCMV. For example, we reported previously that a viral G-protein coupled receptor encoded by GPCMV, GP33, was important for viral pathogenesis in pregnant animals [9]. Schleiss and his collaborators also demonstrated that gp1 encodes macrophage inflammatory protein subfamily of CC chemokine vMIP-1α [10]; gp145 binds dsRNA and antagonizes the protein kinase R pathway [11]; gp147, gp148 and gp149 encode potential MHC-I homologues, and GPCMV lacking the gp147-149 locus replicated with wild-type kinetics in vitro but was essentially avirulent in vivo [12].…”

Time Mating Guinea Pigs by Monitoring Changes to the Vaginal Membrane throughout the Estrus Cycle and with Ultrasound Confirmation

“…Some have described methods in which time mating guinea pigs is achieved by allowing females to carry a pregnancy and then breed in 24 h immediately after birth [ 15 ]. Others simply allow a time range of plus/minus 1 week based on the length at which the female is housed with the male [ 16 ]. However, these methodologies introduce significant variability (mean fetal weight increases from 2.5 ± 0.31 g at GD33 to 12.0 ± 0.77 g at GD39; R. Wilson unpublished) and time which ultimately increases costs associated with performing experiments with guinea pigs.…”