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

Abstract

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.

Treatment of Influenza: Prospects of Post-Transcriptional Gene Silencing Through Synthetic siRNAs

“…94 Sui et al also reported that inhibition of propagation of H1N1 and HPAI H5N1 viruses in stable cell lines by siRNAs targeting the M2 gene was sustained for more than 50 hours post-infection. 79 Similar results were obtained by Ge et al in MDCK cells using siRNA targeting the NP gene (NP-1496). 66…”

“…79 Another study also revealed that siRNAs could effectively reduce the NS1 gene expression of the H5N1 avian influenza virus. 97 Sirnaomics have developed a siRNA-based therapeutic agent against influenza virus, known as STP702.…”

“…In addition, siRNA-resistant mutants did not appear in the targeted sequence, even after culturing the virus for 40 passages. 79 Among the three siRNA expression plasmids designed by Zhou et al against H5N1 avian influenza, siRNA specific for nucleocapsid protein was found to have a specific effect in inhibiting the accumulation of RNAs in infected cells because of a critical requirement for newly synthesized nucleocapsid proteins in avian influenza viral RNA transcription and replication. 99 Tompkins et al also produced broadly effective anti-influenza siRNAs directed against the NP and PA genes.…”

In vivo inhibition of influenza A virus replication by RNA interference targeting the PB2 subunit via intratracheal delivery

“…Ge et al also showed that shRNA, expressed by a lentivirus DNA vector, could prevent influenza A/Puerto Rico/8/34-induced pneumonia in mice. A number of subsequent studies have shown that siRNA targeting different RNA segments can inhibit the replication of highly pathogenic avian influenza virus H5N1 [10, 12, 13] and various influenza A (H1N1) viruses in vitro and in vivo [9, 11, 21–23]. …”

siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus

“…The sequence-specific knockdown of viral genes in infected cells without affecting on host gene expression has generated great interest in development of small interfering RNAs (siRNAs)-based antiviral therapeutics (Elbashir et al 2001;Morris and Rossi 2006). Previous reports have shown that the siRNAs could effectively inhibit IAV replication both in vivo and in vitro (Ge et al 2003;Li et al 2005;Zhou et al 2007;Sui et al 2009;Zhang et al 2009;Li et al 2011).…”