Caspase-mediated cleavage of the feline calicivirus capsid protein.

School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Journal of General Virology (Impact Factor: 3.18). 06/2003; 84(Pt 5):1237-44.
Source: PubMed


Feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the 40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-derived FCV capsid protein, revealed that caspase-2, and to a lesser extent caspase-6, cleaved the capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV infection is discussed.

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Available from: Victoria A Mcguire, Sep 30, 2015
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    • "The activation of caspase 3, together with additional components of the mitochondrial pathway of apoptosis activation , was found also in FCV infected cells (Natoni et al., 2006). Furthermore, an in vitro cleavage assay revealed that caspase-2, as well as caspase-6, cleaved the FCV capsid protein to generate a 40 -kDa fragment suggesting a possible role of capsid cleavage in the life cycle of FCV infection (Al-Molawi et al., 2003). It will be important to verify whether caspase or other cell proteasemediated cleavages are required for replication of the virus, or merely a by-product of apoptosis or necrotic cell death. "
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    ABSTRACT: Murine norovirus, currently the only norovirus that replicates efficiently in tissue culture, has of-fered scientists the first chance to study the entire norovirus life cycle in the laboratory. In addition, the development of reverse genetics for murine norovirus has provided the ideal opportunity for researchers to determine how variation at the genetic level affects pathogenicity in the natural host. Despite differences in the diseases caused by human and murine noroviruses, they possess a significant amount of genetic similarity; hence the general mechanisms of viral genome translation and replication are likely to be highly conserved. Here we aim to summarize our current under-standing of the mechanisms of norovirus transla-tion and replication, highlighting the important role of murine norovirus as a model system in the study of norovirus biology. Introduction Significant advances have been made in the study of human norovirus replication in tissue culture, namely the generation of stable replicon contain-ing cell lines (Chang et al., 2006), the observation that norovirus replication and packaging can be driven in tissue culture (Asanaka et al., 2005; Katayama et al., 2006) and the demonstration that norovirus RNA purified from stool samples is infectious (Guix et al., 2007). However, despite epic efforts (Duizer et al., 2004), a reproducible system to allow the study of the complete human norovirus life cycle remains elusive. Preliminary results have suggested that a highly differentiated tissue culture system may allow human norovirus propagation (Straub et al., 2007), however further studies are required to validate these observa-tions. The identification of murine norovirus (MNV) in 2003 heralded a new era for the study of the basic mechanisms of norovirus translation and replication, as for the first time it provided researchers with a norovirus capable of a full in-fectious cycle in tissue culture (Karst et al., 2003; Wobus et al., 2004). Until the 'missing-link' which will allow human noroviruses to grow efficiently in tissue culture is identified, which recent studies suggest may be at the level of virus entry (Guix et al., 2007), MNV offers the best readily available and easily manipulated norovirus experimental system. In this chapter we aim to summarize how the MNV model has been used to further our understanding of norovirus translation and replication.
    Caliciviruses: Molecular and Cellular Virology, Edited by Grant S. Hansman, Jason Jiang, Kim Yarbrough Green, 06/2010: chapter 11: pages 205; Horizon Scientific Press.
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    • "It has been reported previously that non-caliciviral proteases are capable of processing LC-VP1 as maturation of VP1 and VLP production was observed when LC-VP1 was expressed in CRFK cells and other cell lines when co-infected with Vaccinia virus [44], [45]. It is also possible that the production of an apparent mature capsid cleavage product is the result of cleavage by cellular caspases, induced as the result of multiple transfections, and previously known to process the FCV capsid protein [46]. In addition, we cannot formally exclude the possibility that very low levels of NS6-7 were present due to small amount of genomic RNA not removed during RNase H treatment. "
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    ABSTRACT: Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5' or 3' extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5'end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle. We have demonstrated that PTB interacts with at least two binding sites within the 5'end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication. Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.
    PLoS ONE 03/2010; 5(3):e9562. DOI:10.1371/journal.pone.0009562 · 3.23 Impact Factor
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    • "Viral-induced apoptosis has been demonstrated for a number of viruses including HIV [19], adenovirus [20], hepatitis C virus [21], herpes simplex virus [22], human papillomavirus [23], and influenza virus [24]. Apoptosis, as a feature of calicivirus infection, has recently been characterized by the increase in caspase-3 activity [10,25-27]. It has also been proposed that caspase-3 cleaves the norovirus polyprotein, suggesting that apoptosis might be required for viral replication [28]. "
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    ABSTRACT: Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV) serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.
    Virology Journal 10/2009; 6(1):139. DOI:10.1186/1743-422X-6-139 · 2.18 Impact Factor
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