Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro.

Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur

Porcine sapovirus replication is restricted by the type I interferon response in cell culture.

Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-β resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2',5'-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100-150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host-cell interactions.This research was supported by funding from the Wellcome Trust (Ref: WT097997MA), Biotechnology and Biological Sciences Research Council (Ref: BB/I012303/1) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A2A2A01004292). IG is a Wellcome senior fellow.This is the final version of the article. It first appeared from the Society for General Microbiology via http://dx.doi.org/10.1099/vir.0.071365-

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

UNLABELLED: Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. IMPORTANCE: Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation.This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A2A2A01004292), and by the Wellcome Trust (Ref: WT097997MA). IG is a Wellcome senior fellow. The authors would like to thank Professor Jeong-Sun Kim for providing reagents and critical input into the project.This is the accepted manuscript version of the article. The final version is available from ASM at http://jvi.asm.org/content/early/2014/08/18/JVI.01650-14.abstract

Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production.

Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection. IMPORTANCE: Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E2 (PGE2) pathway. Targeting of COX-1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2-specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We further demonstrate that the production of PGE2 provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be a new target for treatment of sapovirus infection.Wellcome Trust (097997/Z/11/Z); Grant (2014R1A2A2A01004292) of Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning, the Korea Basic Science Institute grant (C33730), and Bio-industry Technology Development Program (315021-04) funded by the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea

Decolonial Exhumation, or the Future Where No One Is Home: Writing Abuse at the Trans-Queer-Feminist Intersection of Tropical Archipelagic Thinking

These poems are taken from an autobiographical book project on same-sex intimate partner abuse entitled SUNNY that interrogates how the conjoint forces of heterosexualism, racial classification, and capitalism—understood as Eurocentric—position the lives of queer people at the margins, or what Maria Lugones calls “coloniality of gender.” In these poems, the future is maternal, “the streaming touch of oil on burnt skin,” hopeful, yet is “still about our body, except only its ruination” or “a hill’s destruction.” Written at the trans-queer-feminist intersection of tropical archipelagic thinking, the future here is a metaphor of “elsewhere” or, in the words of Paul Carter, “a place not here but consisting of many (incommensurable) places reached from here…in the archipelago” (2019, p. 117; 2013). By interweaving a fictionalised version of my own victimisation and that of other victims of gendered violence, this archipelagic poetics of wounding not only carves out a space for a queer narrative of victimisation that is systematically erased and insufficiently represented in mainstream analyses of gender-based violence, but also maps out linkages of grief and solidarity amongst tropical bodies at the margins. In writing these poems, I framed a method of writing called “decolonial exhumation,” which is a creative practice of writing and experimentation that struggles for a narration of the pained and miserable present, but one that does not evade histories of multiple and intersectional oppressions. Inspired by Saidiya Hartman’s concept of “critical fabulation” and also informed by the work of plant evolutionary biologist Banu Subramaniam, decolonial exhumation is a creative mission to develop an epistemology and aesthetics that celebrates the fragmentary, lost, partial, incomplete, and perpetually unrecoverable. It is a trans-queer-feminist political response to the legacies of colonialism and empire, and the durable inequalities that cannot otherwise be fully understood without any reckoning of the colonial past and striving for a tropical future “elsewhere.