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

Abstract

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 calpaincatalysed proteolysis are involved in the HCMV replication process.

CD163ΔSRCR5 MARC-145 Cells Resist PRRSV-2 Infection via Inhibiting Virus Uncoating, Which Requires the Interaction of CD163 With Calpain 1

“…The non-structural 5A phosphoprotein of the hepatitis C virus is an inducer and a substrate of the calciumdependent calpain protease(s) (Kalamvoki and Mavromara, 2004). Calpains also have a role in mediating the proteolytic modification of human cytomegalovirus UL112-113 proteins (Wang et al, 2015). Calpain 1 is required for RNA replication of Echovirus 1 and is especially important at a later stage of infection (Upla et al, 2008).…”

Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis

“…Apart from isoforms derived from differently spliced transcripts, additional protein variants could be generated by posttranslational modifications. In fact, two recent papers reported the detection of 3 small UL112/113 proteins, which were apparently generated by calpain-mediated cleavage (44). In immunoblots using the anti-E1 antiserum, we also detected additional protein bands of low intensity (Fig.…”

Calpain-1: a Novel Antiviral Host Factor Identified in Porcine Small Intestinal Mucus

“…Calpain-1, a calcium-dependent cysteine protease found in almost all eukaryotes, is a heterodimer that consists of a catalytic subunit and regulatory subunit (CAPNS1) ( 36 ). Growing evidence suggests that calpain is associated with viral infection; in particular, its protease activity mediates the proteolytic modification of human cytomegalovirus UL112-113 proteins and enterovirus polyprotein, and calpain-1 interacts with the CD163 receptor to facilitate porcine reproductive and respiratory syndrome virus (PRRSV) uncoating in early endosomes ( 37 , 38 ). Previous studies have described the role of endogenous calpain in assisting and promoting viral replication in host cells, contradicting the protective effect of secretory calpain-1 in porcine small intestinal mucus detected in our study ( 39 , 40 ).…”