Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

Human cytomegalovirus UL76 elicits novel aggresome formation via interaction with S5a of the ubiquitin proteasome system.

HCMV UL76 is a member of a conserved Herpesviridae protein family (Herpes_UL24) that is involved in viral production, latency, and reactivation. UL76 presents as globular aggresomes in the nuclei of transiently transfected cells. Bioinformatic analyses predict that UL76 has a propensity for aggregation and targets cellular proteins implicated in protein folding and ubiquitin-proteasome systems (UPS). Furthermore, fluorescence recovery after photobleaching experiments suggests that UL76 reduces protein mobility in the aggresome, which indicates that UL76 elicits the aggregation of misfolded proteins. Moreover, in the absence of other viral proteins, UL76 interacts with S5a, which is a major receptor of polyubiquitinated proteins for UPS proteolysis via its conserved region and the von Willebrand factor type A (VWA) domain of S5a. We demonstrate that UL76 sequesters polyubiquitinated proteins and S5a to nuclear aggresomes in biological proximity. After knockdown of endogenous S5a by RNA interference techniques, the UL76 level was only minimally affected in transiently expressing cells. However, a significant reduction in the number of cells containing UL76 nuclear aggresomes was observed, which suggests that S5a may play a key role in aggresome formation. Moreover, we show that UL76 interacts with S5a in the late phase of viral infection and that knockdown of S5a hinders the development of both the replication compartment and the aggresome. In this study, we demonstrate that UL76 induces a novel nuclear aggresome, likely by subverting S5a of the UPS. Given that UL76 belongs to a conserved family, this underlying mechanism may be shared by all members of the Herpesviridae.