http://llama.mshri.on.ca/courses/Biophysics205/Papers/Braas_2009.pdf

The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.

/pdf/human-host-factors-required-for-influenza-virus-replication-2bkjr0ptdx.pdf

Human host factors required for influenza virus replication

Influenza A virus has developed strategies to exploit and in some cases subvert cellular proteins and pathways to promote its own replication and to suppress antiviral immune responses. Identification of these host factors would expand the number of potential drug targets far beyond the 11 proteins encoded in the viral genome. Recently, several laboratories have set out to provide an insight into the interface between influenza virus and its host by performing genome-wide siRNA silencing screens to characterize these host proteins and to monitor the effects on viral infectivity. Initial hits from each study were used to search databases of protein–protein interactions, allowing prediction of host-cell pathways likely to be involved either in the viral replicative cycle or in the immune response to viral infection. The results of these screens will promote our understanding of influenza virus biology as well as identify potential targets for the rational design of broad-spectrum antiviral drugs such as siRNA and small molecules.

/pdf/genome-wide-rnai-screen-identifies-human-host-factors-cs6dvig3h7.pdf

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

A Physical and Regulatory Map of Host-Influenza Interactions Reveals Pathways in H1N1 Infection

RNA virus infection is recognized by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm RLRs are comprised of N-terminal caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling In the present study, we generated mice lacking LGP2 and found that LGP2 was required for RIG-I- and MDA5-mediated antiviral responses In particular, LGP2 was essential for type I IFN production in response to picornaviridae infection Overexpression of the CARDs from RIG-I and MDA5 in Lgp2(-/-) fibroblasts activated the IFN-beta promoter, suggesting that LGP2 acts upstream of RIG-I and MDA5 We further examined the role of the LGP2 helicase domain by generating mice harboring a point mutation of Lys-30 to Ala (Lgp2 (K30A/K30A)) that abrogated the LGP2 ATPase activity Lgp2 (K30A/K30A) dendritic cells showed impaired IFN-beta productions in response to various RNA viruses to extents similar to those of Lgp2(-/-) cells Lgp2(-/-) and Lgp2 (K30A/K30A) mice were highly susceptible to encephalomyocarditis virus infection Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain

https://www.pnas.org/content/pnas/107/4/1512.full.pdf

LGP2 is a positive regulator of RIG-I– and MDA5-mediated antiviral responses

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.

/pdf/drosophila-rnai-screen-identifies-host-genes-important-for-2s1vwqefsi.pdf

Drosophila RNAi screen identifies host genes important for influenza virus replication

Tetherin (also known as BST2, CD317 or HM1.24) has recently been reported to inhibit a wide range of viruses. However, the antiviral mechanism of action of tetherin has not been determined. Both ends of the tetherin molecule are associated with the plasma membrane and it forms a homodimer. Therefore, a model in which progeny virions are retained on the cell surface by dimer formation between tetherin molecules on the viral envelope and plasma membrane has been proposed as the antiviral mechanism of action of this molecule. To investigate this possibility, we examined the correlation between dimerization and antiviral activity of tetherin in Lassa and Marburg virus-like particle production systems using tetherin mutants deficient in dimer formation. However, the tetherin mutant with complete loss of dimerization activity still showed apparent antiviral activity, indicating that dimerization of tetherin is not essential for its antiviral activity. This suggests that tetherin retains progeny virions on the cell surface by a mechanism other than dimerization.

/pdf/dimerization-of-tetherin-is-not-essential-for-its-antiviral-5b2nv0ztoj.pdf

Akira Sakurai

Papers

Drosophila RNAi screen identifies host genes important for influenza virus replication

Dimerization of Tetherin Is Not Essential for Its Antiviral Activity against Lassa and Marburg Viruses

Regulation of human T-cell leukemia virus type 1 (HTLV-1) budding by ubiquitin ligase Nedd4.

Identification of host factors involved in influenza virus replication