Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

Abstract

The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.

Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin

“…Like MMPs, cathepsins are able to degrade extracellular matrix proteins. Baculovirus v-cathepsin, however, is not secreted, but accumulates in the endoplasmic reticulum of infected cells as an inactive enzyme where it is retained due to its interaction with v-chitinase (Hodgson et al, 2011, 2013). In AcMNPV, v-cathepsin is only activated and released extracellularly after lysis of infected cells (Hodgson et al, 2011; Hom et al, 2002).…”

Reaching the melting point: Degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination

“…The domain structure of baculovirus chitinases is most similar to that of S. marcescens chitinase A (Henrissat, 1999), which is composed of an N-terminal chitin-binding immunoglobulin-like fold (~500 residues), and a C-terminal domain (~1100 residues) that contains the catalytic site (Hodgson et al, 2013; Young et al, 2005). Baculovirus chitinase N-terminal domains have several conserved aromatic surface residues important for insoluble chitin binding and substrate feeding of chitin chains into the catalytic pocket of the C-terminal α/β barrel (Young et al, 2005).…”

“…These results are consistent amongst different research groups and in different virus-host systems and imply that chitinase binds pro-cathepsin as it folds in the ER. Results from bimolecular fluorescence complementation and pull-down experiments documented that direct association of AcMNPV chitinase and pro-cathepsin does occur in the ER of infected cells (Hodgson et al, 2011, 2013). However, the association of chitinase and pro-cathepsin in cells does not address whether chitinase is a bona fide folding chaperone.…”

“…However, the association of chitinase and pro-cathepsin in cells does not address whether chitinase is a bona fide folding chaperone. Cathepsin undergoes maturation and is active in cells infected with a dual chitinase/cathepsin negative AcMNPV bacmid (Kaba et al, 2004) repaired with cathepsin only (Hodgson et al, 2013). This challenges the hypothesis that chitinase is imperative for pro-cathepsin folding and maturation.…”

“…This challenges the hypothesis that chitinase is imperative for pro-cathepsin folding and maturation. A recombinant virus with a lacZ -inactivated chitinase may still transcribe and translate truncated peptides (Hawtin et al, 1997; Hodgson et al, 2013), which could potentially associate with pro-cathepsin in the ER, affecting pro-cathepsin folding or function.…”