Autographa californica M Nucleopolyhedrovirus ProV-CATH is Activated during Infected Cell Death

Abstract

V-CATH, a cathepsin L-like cysteine protease encoded by the baculovirus Autographa californica M nucleopolyhedrovirus, has been shown to play an essential role in host liquefaction. Similar to cellular cathepsin L, V-CATH is synthesized as an inactive proenzyme and is activated by cleavage of the propeptide. Previous studies indicated that removal of the propeptide was rapid, occurring as soon as the protein could be detected by Western blot, 22 h postinfection. We found, however, that these results reflected artifactual processing of the proenzyme. When the protease inhibitor E-64 was used to prevent this aberration, we found that proV-CATH accumulated in infected cells and activation did not begin until the onset of cell death, at approximately 80 h postinfection. Western blot analysis of fractions of live and dead cells isolated by fluorescence-activated cell sorting revealed that mature V-CATH was found only in dead cells. The regulation of activation of proV-CATH, therefore, was quite different from that of cellular cathepsins. Acridine orange staining revealed that lysosome integrity was lost in dead cells, an occurrence that could lead to the activation of proV-CATH by lysosomal proteases.

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

“…In addition, tunicamycin inhibition of N-linked glycosylation results in aggregation of proV-CATH (Hom & Volkman, 2000). The AcMNPV CHIA has an N-terminal signal peptide and, partly due to its Cterminal KDEL motif, is retained in the endoplasmic reticulum (ER) of infected cells (Thomas et al, 1998;Saville et al, 2002) until released upon cell death (Hom et al, 2002).…”

“…Presumably, proV-CATH enters the secretory route and becomes glycosylated, but is not secreted from cells, despite the absence of any recognized retention mechanisms. Rather, the proenzyme is retained in infected cells until cell death occurs, concomitant with V-CATH catalytic maturation (Hom et al, 2002).…”

“…Protein samples from cell lysates, treated with the cysteine protease inhibitor E64, were taken from CfMNPV-infected Cf 203 cells and AcMNPV (or bacmid)-infected Sf 21 cells at 72 and 40 h p.i., respectively. Proteins were analysed by Western blotting using either anti-V-CATH antiserum as described by Hom et al (2002) or anti-HA antibody (Sigma) as per the manufacturer's instructions. Proteins were detected on an X-ray film using the SuperSignal chemiluminescent system (Pierce), and molecular masses were based on PAGE Ruler standards (Fermentas).…”

Recent Advances in Our Knowledge of Baculovirus Molecular Biology and Its Relevance for the Registration of Baculovirus-Based Products for Insect Pest Population Control

“…Horizontal transmission to neighboring larvae is dependent upon the release of OBs that occurs following larval death. Expression of viral proteases (cathepsin) and chitinase, late post infection, ensures that, in most alphabaculoviruses and in some betabaculoviruses, progeny OBs are released in the environment by lysing larval tissues and the exoskeleton of larvae following death (Hawtin et al, 1997;Hom et al, 2002). In addition, in silkmoth and gypsy moth, virally-produced proteins, tyrosine phosphatase (ptp) (Kamita et al, 2005) and ecdysteroid uridine 5´-diphosphate (UDP)-glucosyltransferase (egt) (Hoover et al, 2011), are responsible for behavioral changes that occur during the infection process where infected larvae leave their normal sheltered habitats and climb to exposed surfaces.…”

Improvement of GFPuv-β3GnT2 Fusion Protein Production by Suppressing Protease in Baculovirus Expression System

“…12,13) When insect cells are infected with baculoviruses, the V-CATH accumulates as a propeptide, proV-CATH in infected cells, and the death of infected cells provokes activation of the V-CATH. 14) Bombyx mori nuclear polyhedrosis virus (BmNPV) also has a V-CATH-like cysteine protease. 15) To prevent proteolytic degradations of expressed proteins, cysteine protease-deˆcient viruses were constructed.…”