Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.

[1]  D. Guay,et al.  Amantadine-resistant influenza A in a nursing facility. , 1992, Archives of internal medicine.

[2]  Eugene A. Davidson.  RNA INTERFERENCE TECHNOLOGY: FROM BASIC SCIENCE TO DRUG DEVELOPMENT , 2005 .

[3]  Ben Berkhout,et al.  HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome , 2005, Nucleic acids research.

[4]  F. Graham,et al.  Immunogenicity in mice of tandem repeats of an epitope from herpes simplex gD protein when expressed by recombinant adenovirus vectors. , 1993, Vaccine.

[5]  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.

[6]  Angus Nicoll,et al.  Avian influenza A (H5N1) infection in humans. , 2005, The New England journal of medicine.

[7]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[8]  John F. G. Atack,et al.  RNA Interference , 2010, Methods in Molecular Biology.

[9]  M. Manns,et al.  Caspase 8 small interfering RNA prevents acute liver failure in mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Qing Ge,et al.  Inhibition of influenza virus production in virus-infected mice by RNA interference. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E. Kruithof,et al.  Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors , 2004, BMC Molecular Biology.

[12]  Kazuhiko Hayashi,et al.  siRNA‐resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations , 2006, Journal of viral hepatitis.

[13]  Ben Berkhout,et al.  Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition , 2004, Journal of Virology.

[14]  N. Zhong,et al.  Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque , 2005, Nature Medicine.

[15]  Constance Schultsz,et al.  Avian influenza A (H5N1) in 10 patients in Vietnam. , 2004, The New England journal of medicine.

[16]  J. Sham,et al.  Anti-tumor effects of human peripheral gammadelta T cells in a mouse tumor model. , 2001, International journal of cancer.

[17]  A. Ganser,et al.  Specific inhibition of bcr-abl gene expression by small interfering RNA. , 2003, Blood.

[18]  B. Cullen,et al.  Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors , 2005, The journal of gene medicine.

[19]  H. Vornlocher,et al.  Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). , 2003, Blood.

[20]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[21]  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.

[22]  Michael G. Katze,et al.  Viruses and interferon: a fight for supremacy , 2002, Nature Reviews Immunology.

[23]  Samson S. Y. Wong,et al.  Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus , 2008, Proceedings of the National Academy of Sciences.

[24]  Yaochen Li,et al.  Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus. , 2005, Avian diseases.

[25]  A. García-Sastre.  Mechanisms of inhibition of the host interferon α/β-mediated antiviral responses by viruses , 2002 .

[26]  Yi Guan,et al.  Three Indonesian clusters of H5N1 virus infection in 2005. , 2006, The New England journal of medicine.

[27]  R. Krug,et al.  Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[28]  K. Chan,et al.  Anti‐tumor effects of human peripheral γδ T cells in a mouse tumor model , 2001 .

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

[30]  I. Chen,et al.  Efficient lentiviral vectors for short hairpin RNA delivery into human cells. , 2003, Human gene therapy.

[31]  R. Webster,et al.  Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice. , 2005, The Journal of infectious diseases.

[32]  R. Krug,et al.  Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer , 1988, Journal of virology.

[33]  Kaoru Saigo,et al.  Short-Interfering-RNA-Mediated Gene Silencing in Mammalian Cells Requires Dicer and eIF2C Translation Initiation Factors , 2003, Current Biology.

[34]  J. Stockman,et al.  Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China , 2010 .

[35]  M. Wiznerowicz,et al.  A versatile tool for conditional gene expression and knockdown , 2006, Nature Methods.

[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.

[37]  R. Webster,et al.  Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en , 1997, Journal of virology.

[38]  Prasert Auewarakul,et al.  Probable person-to-person transmission of avian influenza A (H5N1). , 2005, The New England journal of medicine.

[39]  N. Cox,et al.  Amantadine-resistant influenza A in nursing homes. Identification of a resistant virus prior to drug use. , 1995, Archives of internal medicine.

[40]  A. García-Sastre.  Mechanisms of inhibition of the host interferon alpha/beta-mediated antiviral responses by viruses. , 2002, Microbes and infection.

[41]  Judy Lieberman,et al.  RNA interference targeting Fas protects mice from fulminant hepatitis , 2003, Nature Medicine.

[42]  Madhur Kumar,et al.  Antisense RNA: Function and Fate of Duplex RNA in Cells of Higher Eukaryotes , 1998, Microbiology and Molecular Biology Reviews.

[43]  M. Wiznerowicz,et al.  Lentivirus-mediated Rna Interference of Dc-sign Expression Inhibits Human Immunodeficiency Virus Transmission from Dendritic Cells to T Cells in the Early Events of Human Immunodeficiency Virus Type 1 (hiv-1) Infection, Immature Dendritic Cells (dcs) Expressing the Dc-specific Intercellular Adhesion , 2022 .

[44]  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.