Autographa californica multiple nucleopolyhedrovirus and Choristoneura fumiferana multiple nucleopolyhedrovirus v-cath genes are expressed as pre-proenzymes

Abstract

Intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH), which lacks canonical targeting signals, are poorly understood. The cathepsins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and most other alphabaculovirus group I nucleopolyhedroviruses have well-conserved N-termini containing overlapping chymotrypsin-cleavage (Y11) and myristoylation (G12) motifs, which are suggestive of proteolytic signal-peptide cleavage to generate proV-CATH and subsequent acylation. To determine proteolytic N-terminal processing of V-CATH, haemagglutinin epitope-coding tags were fused to the 5′ and/or 3′ ends of AcMNPV and CfMNPV v-cath. Immunoblot analysis suggested that a small N-terminal peptide is cleaved for both viruses, indicating that v-cath is expressed as a pre-proenzyme. The two viral homologues undergo similar proteolytic processing, but have different glycosylation or other post-translational modifications. An AcMNPV V-CATH–DsRED fusion protein co-localized to the endoplasmic reticulum with an HDEL motif-containing green fluorescent protein. Based on these findings, pre-proV-CATH processing and trafficking mechanisms are postulated.

Interaction of Autographa californica Multiple Nucleopolyhedrovirus Cathepsin Protease Progenitor (proV-CATH) with Insect Baculovirus Chitinase as a Mechanism for proV-CATH Cellular Retention

“…Monolayers of Sf21 or Hi5 cells were grown in Grace's insect medium supplemented with fetal bovine serum and penicillin-streptomycin as described previously (13). Cell monolayers were inoculated with AcMNPV at a multiplicity of infection (MOI) of 10 (unless otherwise specified) and were incubated for 1 h at room temperature, after which the inoculum was removed and replaced with growth medium.…”

“…S1a in the supplemental material). Since v-cath naturally contains an ApaI site, the KpnI-FLAG-EcoRV cassette was subcloned by use of KpnI/EcoRV into pchiA.cathHA (13) to generate pCH/CA (see Fig. S1a).…”

“…If v-cath is not expressed or if proV-CATH is rendered insoluble due to chiA deletion, cell lysis is reduced, as measured in vitro by reduced polyhedron release or in vivo by reduced polyhedron content and turbidity of the hemolymph and a lack of liquefaction (11,26,29). In addition, glycosylation of AcMNPV, Bombyx mori NPV (BmNPV), and Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) proV-CATH is required for its proper folding and/or maintenance in a soluble form (13,15,21). proV-CATH expressed without N-linked glycans, due to tunicamycin inhibition (15,26) or the substitution of alanine for asparagine acceptor residues (19), formed insoluble aggregates, and consequently, mature, enzymatically active V-CATH was not produced.…”

“…The signal peptide of the nascent polypeptide is removed upon cotranslational import into the ER, where the polypeptide is posttranslationally modified at Asn65 with an N-linked glycan (15,26). It has been postulated that the secretory signal peptide of preproV-CATH might be cleaved after Tyr11, unmasking a potential myristate lipid conjugation motif and a requisite Nterminal Gly12 myristate acceptor (13). The putative myristoylation of proV-CATH at Gly12 would offer a mechanism by which proenzyme secretion might be blocked, due to a hydrophobic interaction of myristic lipid with cellular lipid membranes or with viral CHIA or other ER proteins.…”

“…AcMNPV v-cath is initially translated as a preproenzyme (13). The signal peptide of the nascent polypeptide is removed upon cotranslational import into the ER, where the polypeptide is posttranslationally modified at Asn65 with an N-linked glycan (15,26).…”