Functional analysis of human cytomegalovirus pUS28 mutants in infected cells

Abstract

The human cytomegalovirus (HCMV)-encoded viral G protein-coupled receptor pUS28 contributes to an array of biological effects, including cell migration and proliferation. Using FIX-BAC (bacterial artificial chromosome, derived from the HCMV clinical isolate VR1814) and lambda red recombination techniques, we generated HCMV recombinants expressing amino-terminally FLAG-tagged versions of wild-type pUS28 (FLAG–US28/WT), G-protein coupling deficient pUS28 (FLAG–US28/R129A) and chemokine-binding domain deficient pUS28 (FLAG–US28/ΔN). Infection with the FLAG–US28/R129A virus failed to induce inositol phosphate accumulation, indicating that G-protein coupling is essential for pUS28 signalling to phospholipase C-β (PLC-β) during HCMV infection. The FLAG–US28/ΔN virus induced about 80 % of the level of PLC-β signalling induced by the FLAG–US28/WT virus, demonstrating that the N-terminal chemokine-binding domain is not required for pUS28-induced PLC-β signalling in infected cells. The data presented here are the first to describe the functional analyses of several key pUS28 mutants in HCMV-infected cells. Elucidating the mechanisms by which pUS28 signals during infection will provide important insights into HCMV pathogenesis.

The Carboxy-Terminal Tail of Human Cytomegalovirus (HCMV) US28 Regulates both Chemokine-Independent and Chemokine-Dependent Signaling in HCMV-Infected Cells

“…Bacterial artificial chromosomes (BACs) containing either the wild-type VR1814 strain of HCMV (FIX-BAC) or a mutant in which the US28 open reading frame was replaced by a Kan r /LacZ cassette (FIX-BAC-⌬US28) have been previously described (16,49). Lambda red recombination was utilized to construct a mutant of US28 termed US28/1-314 from which amino acids 315 to 354 have been deleted.…”

Cell-type specific activities of cytomegalovirus GPCR homologues

“…35kDa). Although counter-intuitive, this result is consistent with published data suggesting that the deleted region of the N-terminus may be a site of post-translational modification, possibly O-linked glycosylation Stropes & Miller, 2008). Data from B105 sequencing confirmed the correct sequence, whereas B182 lacked the deletion.…”

“…For example, M33 of mouse CMV has a variant NRY motif at this position; N-D mutation resulted in enhanced signalling, whereas R-Q resulted in ablation of signalling (Case et al, 2008). Similarly, an R-A mutation on US28 resulted in a signalling deficient phenotype (Stropes & Miller, 2008). Here, the D128N substitution, which retained >70% of CREB activation levels compared with wt UL33, did not affect migration in contrast to the signalling deficient R129Q mutation.…”

“…US28 also stimulates the PLC-β pathway through a GPCR ligand-dependent manner. The disruption of the DRY motif (for a DAY motif) not only ablates PLC-β signalling, but also diminishes CCL5 binding ability -however not due to interference with surface expression -, demonstrating the importance of this region for both ligand-dependent and independent signalling (Stropes & Miller, 2008). Curiously, the signalling profile and desensitization mechanisms of US28 and M33 are closer to each other than M33 and its human homolog UL33 and US28 can also partially rescue M33 phenotype (Farrell et al, 2013;Sherrill & Miller, 2006;Waldhoer et al, 2002).…”

“…G protein-coupling was confirmed to play a key role in this process as disruption of the GPCR DRY-motif prevented IP accumulation following HCMV infection of fibroblasts (Stropes & Miller, 2008).…”