Biology subjects

Hosmillo, M.

Publications and source records attributed to Hosmillo, M..

4 recordsLinked to original sources

bioRxiv2019

Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells.

BackgroundNorovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins, which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5 terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5 end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery.\n\nMethodsIfit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro.\n\nResultsHere, we show that VPg-dependent translation is completely refractory Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5 end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling.\n\nConclusionsIfit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.

immunology↗

bioRxiv2019

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.

microbiology↗

bioRxiv2019

A robust human norovirus replication model in zebrafish larvae

Human noroviruses (HuNoVs) are an important cause of epidemic and endemic acute gastroenteritis worldwide; annually about 700 million people develop a HuNoV infection resulting in [~]219,000 deaths and a societal cost estimated at 60 billion US dollars 1. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics against HuNoV. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. HuNoV is detected in cells of the hematopoietic lineage, the intestine, liver and pancreas. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Downregulation of fucosyltransferase 8 (fut8) in the larvae reduces HuNoV replication, highlighting a common feature with infection in humans. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

microbiology↗

bioRxiv2019

Epigenetic suppression of interferon lambda receptor expression leads to an enhanced HuNoV replication in vitro

Human norovirus (HuNoV) is the main cause of acute gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in epithelial human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring the replication of viral RNA. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-{beta} by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-{lambda}. Inhibition of DNA methyltransferase activity with 5-aza-2-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this observation, ectopic expression of the IFN-{lambda} receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-{lambda}. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. To the best of our knowledge, this study unravels for the first time epigenetic reprograming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication.

microbiology↗