Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells

Significance 

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an Alphabaculovirus from the Baculoviridae family. Baculoviruses possess large double-stranded DNA and mainly infect lepidopteran insects. Baculovirus-infected larvae typically release progeny viral occlusion bodies into the surrounding environment, which allows for the cyclical transmission of the virus to new hosts. Virus-induced larval liquefaction is a characteristic feature of terminal alphabaculovirus-infected cadavers that is needed for the efficient dissemination of occlusion bodies. The baculovirus chitinase A-like (chiA) and viral cathepsin (v-cath) are classified as auxiliary genes because upon deletion of either or both genes baculoviruses tend to be just as lethal to host larvae as viruses containing chiA/v-cath. The co-expression and co-regulation of alphabaculovirus chitinase (CHIA) and viral cathepsin (V-CATH) enzymes are during infection fosters terminal liquefaction of host insect cadavers, thereby facilitating the dispersal of occlusion bodies. However, there has been no detailed investigation into the individual roles that both CHIA and V-CATH play in promoting virus-induced cell lysis that is a prerequisite for enzymatic CHIA/V-CATH dissolution of host insect tissues and dissemination of occlusion bodies.

In a new study published in Virology, Dr. Jeffrey Hodgson from the Boyce Thompson Institute at Cornell University together with Dr. Peter Krell at University of Guelph, and Dr. A. Lorena Passarelli at Kansas State University examined the effects v-cath expression had on the cellular release of CHIA, V-CATH and occlusion bodies. The authors showed that V-CATH expression and proteolytic cleavage to its active form was essential for optimal cell lysis and the release of CHIA, V-CATH and progeny occlusion bodies into both the hemolymph of insects infected with AcMNPV and the medium of infected cultured cells.

The research team did not find any evidence of lysis in cells infected with viruses that did not express v-cath. This was demonstrated by the lack of occlusion bodies released into the hemolymph of infected insects or the extracellular growth medium of cultured cells. Following the treatment of infected cells throughout infection with E-64, an irreversible inhibitor of cysteine protease activity, it was observed that proV-CATH was not processed into V-CATH, the activity levels of V-CATH were reduced, and occlusion bodies were not released from cells. This indicated that the lysis of cultured cells required the maturation and/or activity of V-CATH. The native levels of v-cath expression were found to be sufficient at promoting the release from occlusion bodies from cells and the production of processed V-CATH correlated with the release of OBs from cells.

The overexpression of v-cath in infected cells increased V-CATH activity at 48 hours post infection although release of occlusion bodies from the cultured cells were at levels similar to when v-cath was expressed from its native promoter. Contrastingly, v-cath overexpression led to the enhancement of the release of CHIA, V-CATH and occlusion bodies into the hemolymph of infected insects at 3 days post infection. The release of CHIA into hemolymph did not absolutely require v-cath expression, but efficient cellular release of CHIA required the co-expression of v-cath. The overexpression of v-cath however, did not significantly facilitate increased cellular release of CHIA, V-CATH or occlusion bodies. They also showed that the activity and maturation of AcMNPV V-CATH and the native level of both chiA and v-cath enable the release of CHIA and V-CATH and progeny occlusion bodies from infected cells, which ultimately regulates both host liquefaction and therefore efficient dispersal of occlusion bodies.

Through a series of elegant molecular and functional experiments, the authors suggested the presence of an important link between V-CATH activity and the cellular release of CHIA, V-CATH and progeny occlusion bodies from cells.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells - Medicine Innovates
Figure 1: Pathology of Trichoplusia ni larvae at 4 days post-infection. Third instar larvae (from Benzon Research) were injected with 5 x 104 TCID50 units (in 10 µl) of the budded form of each virus and placed in individual 5 ounce plastic cups containing artificial insect diet (Southland) in an incubator at 27 °C with 12:12 light:dark cycle. At 4 days post-infection insects were photographed with an Olympus camera. Viruses used were: AcEGFP (GFP) which lacks both chiA and v-cath, AcWTrepair (WT) that expresses chiA and v-cath from their native promoters, Acp6.9-chiA (p6.9) which overexpresses chiA from the AcMNPV p6.9 promoter but does not express v-cath, Acp6.9-chiA/polh-cath (p6.9/polh) which overexpresses chiA from the p6.9 promoter and v-cath from the AcMNPV polh promoter. Note that only the p6.9/polh virus infected larva is liquefying at this timepoint.

About the author

Jeffrey Hodgson graduated with a B.Sc from the University of Waterloo and obtained a Ph.D degree from the lab of Dr. Peter Krell in the Dept. of Molecular and Cellular Biology at the University of Guelph, where he studied the co-regulated subcellular trafficking and cellular release of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV; an alphabaculovirus) chitinase (CHIA) and cathepsin (V-CATH) enzymes. His publications based on this work describe the subcellular trafficking of the inactive V-CATH enzyme progenitor (proV-CATH) and its molecular interaction with CHIA in the endoplasmic reticulum (ER) of infected cells. He found that the ER retention “KDEL” motif of CHIA was required to sequester proV-CATH in the ER as a CHIA/proV-CATH complex, and that deletion of the CHIA KDEL resulted in the constitutive co-secretion of CHIA and proV-CATH. This research also documented that AcMNPV CHIA is not required as a folding chaperone for nascent proV-CATH despite contrasting reports of mis-folded proV-CATH from three independent laboratories that each generated and studied chiA-null alphabaculoviruses. His recent publication “Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells” follows on his Ph.D work and highlights the importance of the how these viral enzymes are regulated at the cellular level in order to promote environmental dissemination of progeny virions.

The focus of Dr. Hodgson’s current research at The Boyce Thompson Institute is elucidating the subcellular trafficking route(s) of envelope fusion proteins from AcMNPV and from arboviruses (Dengue, vesicular stomatitis virus) in both cultured insect cells and cells of the insect gut and salivary gland. Host cell protein trafficking pathways are needed to transport viral envelope proteins – integral membrane proteins – to the subcellular site where assembly of progeny virus particles occur. The directional, polarized transport of the viral envelope proteins in infected vector insect (i.e. mosquito) gut or salivary gland cells enables transmission of infectious virus particles from infected mosquitoes (or other biting insects) to humans, livestock or plant hosts in which they often cause disease. Overall, due to many years of studying subcellular viral protein trafficking in insects, Dr. Hodgson’s work is anticipated to yield valuable information for managing virus transmission by insects.

Reference

Hodgson JJ, Krell PJ, Passarelli AL. Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells. Virology. 2021;556:23-32.

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