Inhibition of influenza A virus matrix and nonstructural gene expression using RNA interference.

Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influenza virus therapy.

[1]  Li Yao-chen,et al.  Construction of Influenza Virus siRNA Expression Vectors and Their Inhibitory Effects on Multiplication of Influenza Virus , 2005 .

[2]  Y. Bryson,et al.  Double-blind controlled study of central nervous system side effects of amantadine, rimantadine, and chlorpheniramine , 1982, Antimicrobial Agents and Chemotherapy.

[3]  Michael T. McManus,et al.  RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[5]  R. Lamb,et al.  Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface , 1985, Cell.

[6]  P. Palese,et al.  The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins , 1998, The EMBO journal.

[7]  Adolfo García-Sastre,et al.  Induction and evasion of type I interferon responses by influenza viruses. , 2011, Virus research.

[8]  R. Reichman,et al.  A controlled trial of amantadine and rimantadine in the prophylaxis of influenza A infection. , 1982, The New England journal of medicine.

[9]  A. Pichlmair,et al.  RIG-I-Mediated Antiviral Responses to Single-Stranded RNA Bearing 5'-Phosphates , 2006, Science.

[10]  A. Wilcox,et al.  Risk of fetal death after pandemic influenza virus infection or vaccination. , 2013, The New England journal of medicine.

[11]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[12]  J. Mcswiggen,et al.  A potential treatment for pandemic influenza using siRNAs targeting conserved regions of influenza A , 2008 .

[13]  R. Lamb,et al.  Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block , 1993, Journal of virology.

[14]  A. Schmidt.  Antiviral Therapy for Influenza , 2012, Drugs.

[15]  J. Drake.  Rates of spontaneous mutation among RNA viruses. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[16]  E. Akl,et al.  Antivirals for Treatment of Influenza , 2012, Annals of Internal Medicine.

[17]  Tomoyuki Yamada,et al.  Accelerated off-target search algorithm for siRNA , 2005, Bioinform..

[18]  I. Chen,et al.  Inhibition of influenza virus matrix (M1) protein expression and virus replication by U6 promoter-driven and lentivirus-mediated delivery of siRNA. , 2004, The Journal of general virology.

[19]  Keiji Fukuda,et al.  Influenza-associated hospitalizations in the United States. , 2004, JAMA.

[20]  A. Hay,et al.  Regulation of pH by the M2 protein of influenza A viruses. , 1992, Virus research.

[21]  Laurent Kaiser,et al.  Influenza virus neuraminidase inhibitors , 2000, The Lancet.

[22]  Hakim Tafer,et al.  Bioinformatics of siRNA design. , 2014, Methods in molecular biology.

[23]  D. Leake,et al.  Development of therapeutic siRNAs for pachyonychia congenita. , 2008, The Journal of investigative dermatology.

[24]  K. Ungchusak,et al.  Severe Human Influenza Infections in Thailand: Oseltamivir Treatment and Risk Factors for Fatal Outcome , 2009, PloS one.

[25]  D. Beezhold,et al.  Enhanced detection of infectious airborne influenza virus. , 2011, Journal of virological methods.

[26]  K. Lohman,et al.  Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes , 2002, Journal of Clinical Microbiology.

[27]  E. Hirayama,et al.  Nuclear export of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation of viral matrix protein. , 2003, Virology.

[28]  O. Haller,et al.  Mx-dependent resistance to influenza viruses is induced by mouse interferons α and β but not γ , 1984 .

[29]  Jian-Dong Huang,et al.  Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication , 2009, PloS one.

[30]  M. Biggerstaff,et al.  H1N1 2009 influenza virus infection during pregnancy in the USA , 2009, The Lancet.

[31]  S. Kauppinen,et al.  Treatment of HCV infection by targeting microRNA. , 2013, The New England journal of medicine.

[32]  Larisa V. Gubareva,et al.  Surveillance for Neuraminidase Inhibitor Resistance among Human Influenza A and B Viruses Circulating Worldwide from 2004 to 2008 , 2008, Antimicrobial Agents and Chemotherapy.

[33]  Meilin Jin,et al.  Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice. , 2007, Antiviral research.

[34]  A. Monto,et al.  Clinical signs and symptoms predicting influenza infection. , 2000, Archives of internal medicine.

[35]  R. Ruigrok,et al.  Electron microscopy of the influenza virus submembranal structure. , 1989, Virology.

[36]  T. Tumpey,et al.  Protection against lethal influenza virus challenge by RNA interference in vivo. , 2004, Proceedings of the National Academy of Sciences of the United States of America.