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

Influenza A viruses are zoonotic pathogens that continuously circulate and change in several animal hosts, including birds, pigs, horses and humans. The emergence of novel virus strains that are capable of causing human epidemics or pandemics is a serious possibility. Here, we discuss the value of surveillance and characterization of naturally occurring influenza viruses, and review the impact that new developments in the laboratory have had on our understanding of the host tropism and virulence of viruses. We also revise the lessons that have been learnt from the pandemic viruses of the past 100 years.

Influenza A viruses: new research developments

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

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

L'invention concerne des procedes d'amplification, de detection, de mesure, et d'identification des ARNmi a partir d'echantillons biologiques, en particulier des quantites limitees d'un echantillon biologique. Les ARNmi qui sont de maniere differentielle exprimes dans des echantillons de tumeur et des tissus normaux sont utiles en tant que biomarqueurs de cancer pour le diagnostic d'un cancer.

Réactifs et procédés permettant une analyse d'expression d'arnmi et identification de biomarqueurs de cancer

L'invention concerne des cellules de cancer cervicales et des cellules de cancer de la tete ou du cou « HPV » qui expriment trois genes specifiques des testicules non exprimes normalement dans des cellules somatiques; une molecule d'adherence cellulaire testiculaire 1 (TCAM1), une proteine de complexe synaptosomal 2 (SYCP2) et un antigene stromal 3 (STAG3) Parmi les trois marqueurs, 'TCAM1 et SYCP2 sont des marqueurs de detection precoces Divers procedes pour identifier un etre humain ou un animal non humain en tant que candidat pour un examen supplementaire pour le cancer cervical, une lesion preneoplasique pour un cancer cervical, un cancer de la tete et du cou ou une lesion preneoplasique pour un cancer de la tete et du cou sont reveles Des procedes de detection desdits cancers et des lesions preneoplasiques, des procedes de criblage de medicament pour traiter lesdits cancers et lesdites lesions preneoplasiques, des procedes pour surveiller l'efficacite d'un traitement pour lesdits cancers et des procedes pour traiter lesdits cancers sont egalement reveles L'invention concerne en outre des kits qui peuvent etre utilises pour mettre en pratique les procedes ci-dessus

Paul G. Ahlquist

Papers

Drosophila RNAi screen identifies host genes important for influenza virus replication

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

Réactifs et procédés permettant une analyse d'expression d'arnmi et identification de biomarqueurs de cancer

Biomarqueurs pour cancers associés au papillomavirus humain