Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins

2013

J Virol

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.

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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

2013

J Virol

“…These pFastBAC vectors were used to integrate the various gene constructs into the bacmid genome by using standard bacmid technology to generate the corresponding recombinant AcBAC⌬CC-derived AcMNPVs, as summarized in Table 1. The AcBAC⌬CC bacmid had its native chiA-v-cath locus deleted (18), so that the various chiA and v-cath gene constructs were located only in the bacmid polh locus engineered into AcBAC⌬CC and were expressed under the control of their native intergenic promoters by these viruses. The integrity of all pFastBAC clones was verified by DNA sequencing, and that of the corresponding AcBAC⌬CC-derived viral constructs was confirmed by PCR.…”

Section: Cells and Virusmentioning

confidence: 99%

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

2011

J Virol

The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA-proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA-green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA-proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA-proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ⌬KDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.Baculovirus infection is initiated after the ingestion of food contaminated with occlusion bodies (OBs) containing the infectious enveloped virions. OBs dissolve in the insect midgut and release the embedded virions (7). Baculoviruses in the Alphabaculovirus and Betabaculovirus genera cause systemic infections in their lepidopteran hosts, and progeny OBs are disseminated only after the death of the infected larvae. The release of alpha-and betabaculovirus OBs from the infected larval cadaver into the environment is enabled by the tightly regulated release and activation of the virus-encoded chitinase (CHIA) and cathepsin (V-CATH) enzymes, which act in concert to liquefy the host carcass. The subcellular retention, release, and activation of these two enzymes are coordinated to allow for dissolution only after optimal virus production (11,21,26). Once the host succumbs to the virus infection, viral OBs are liberated from the insect cadaver to facilitate horizontal transmission to other larvae. Most alpha-and betabaculoviruses contain homologues of chiA and v-cath, and several of ...

“…2a). The native contiguous and divergent orientation of chiA to v-cath and of the chiA/v-cath intergenic promoter locus were preserved to allow expression from the native promoters, but the two open reading frames (ORFs) were cloned into the polh locus of a bacmid lacking the native chiA and v-cath ORFs (Kaba et al, 2004). Thus, the engineered chiA/v-cath expression constructs were studied in the context of an otherwise normal virus infection.…”

mentioning

confidence: 99%

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

Journal of General Virology

2009

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.