Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1

Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN- dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING- dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals

PLoS Pathog

Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes

Hepatitis C virus infection protein network

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein–protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFβ pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins

The low-density lipoprotein receptor and apolipoprotein E associated with CCHFV particles mediate CCHFV entry into cells

The Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging pathogen of the Orthonairovirus genus that can cause severe and often lethal hemorrhagic diseases in humans. CCHFV has a broad tropism and can infect a variety of species and tissues. Here, by using gene silencing, blocking antibodies or soluble receptor fragments, we identify the low-density lipoprotein receptor (LDL-R) as a CCHFV entry factor. The LDL-R facilitates binding of CCHFV particles but does not allow entry of Hazara virus (HAZV), another member of the genus. In addition, we show that apolipoprotein E (apoE), an exchangeable protein that mediates LDL/LDL-R interaction, is incorporated on CCHFV particles, though not on HAZV particles, and enhances their specific infectivity by promoting an LDL-R dependent entry. Finally, we show that molecules that decrease LDL-R from the surface of target cells could inhibit CCHFV infection. Our study highlights that CCHFV takes advantage of a lipoprotein receptor and recruits its natural ligand to promote entry into cells

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such ‘ORFeome’ resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway® system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins

Field site selection: getting it right first time around

The selection of suitable field sites for integrated control of Anopheles mosquitoes using the sterile insect technique (SIT) requires consideration of the full gamut of factors facing most proposed control strategies, but four criteria identify an ideal site: 1) a single malaria vector, 2) an unstructured, relatively low density target population, 3) isolation of the target population and 4) actual or potential malaria incidence. Such a site can exist in a diverse range of situations or can be created. Two contrasting SIT field sites are examined here: the desert-flanked Dongola Reach of the Nile River in Northern State, Sudan, where malaria is endemic, and the island of La Reunion, where autochthonous malaria is rare but risk is persistent. The single malaria-transmitting vector at both sites is Anopheles arabiensis. In Sudan, the target area is a narrow 500 km corridor stretching from the rocky terrain at the Fourth Cataract - just above the new Merowe Dam, to the northernmost edge of the species range, close to Egypt. Vector distribution and temporal changes in density depend on the Nile level, ambient temperature and human activities. On La Reunion, the An. arabiensis population is coastal, limited and divided into three areas by altitude and exposure to the trade winds on the east coast. Mosquito vectors for other diseases are an issue at both sites, but of primary importance on La Reunion due to the recent chikungunya epidemic. The similarities and differences between these two sites in terms of suitability are discussed in the context of area-wide integrated vector management incorporating the SIT

IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity

Alternative Ii-independent antigen-processing pathway in leukemic blasts involves TAP-dependent peptide loading of HLA class II complexes

During HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. This study explores an alternative HLA class II presentation pathway in leukemic blasts that involves proteasome and transporter associated with antigen processing (TAP)-dependent peptide loading. Although HLA-DR did associate with Ii, Ii silencing in the human class II-associated invariant chain peptide (CLIP)-negative KG-1 myeloid leukemic cell line did not affect total and plasma membrane expression levels of HLA-DR, as determined by western blotting and flow cytometry. Since HLA-DR expression does require peptide binding, we examined the role of endogenous antigen-processing machinery in HLA-DR presentation by CLIP− leukemic blasts. The suppression of proteasome and TAP function using various inhibitors resulted in decreased HLA-DR levels in both CLIP− KG-1 and ME-1 blasts. Simultaneous inhibition of TAP and Ii completely down-modulated the expression of HLA-DR, demonstrating that together these molecules form the key mediators of HLA class II antigen presentation in leukemic blasts. By the use of a proteasome- and TAP-dependent pathway for HLA class II antigen presentation, CLIP− leukemic blasts might be able to present a broad range of endogenous leukemia-associated peptides via HLA class II to activate leukemia-specific CD4+ T cells