5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

Limited protection of current vaccines and antiviral drugs against influenza A virus infection underscores the urgent need for development of novel anti-influenza virus interventions. While short interfering RNA (siRNA) has been shown to be able to inhibit influenza virus infection in a gene-specific manner, activation of the retinoic acid-inducible gene I protein (RIG-I) pathway has an antiviral effect in a non-gene-specific mode. In this study, we designed and tested the anti-influenza virus effect of a short double-stranded RNA, designated 3p-mNP1496-siRNA, that possesses dual functions: an siRNA-targeting influenza NP gene and an agonist for RIG-I activation. This double-stranded siRNA possesses a triphosphate group at the 5= end of the sense strand and is blunt ended. Our study showed that 3p-mNP1496-siRNA could potently inhibit influenza A virus infection both in cell culture and in mice. The strong inhibition effect was attributed to its siRNA function as well as its ability to activate the RIG-I pathway. To the best of our knowledge, this is the first report that the combination of siRNA and RIG-I pathway activation can synergistically inhibit influenza A virus infection. The development of such dual functional RNA molecules will greatly contribute to the arsenal of tools to combat not only influenza viruses but also other important viral pathogens.

Section: Discussionmentioning

confidence: 99%

5′-Triphosphate-Short Interfering RNA: Potent Inhibition of Influenza A Virus Infection by Gene Silencing and RIG-I Activation

Lin

Liu

Bérubé

et al. 2012

“…However, in this context, a recently discovered RLR pathway that comprises a cytosolic class of receptors, such as RIG-I, specialized in sensing viral nucleic acids (36), has not been well explored, particularly the effects of RIG-I pathway activation in reducing vaccine antigen doses in the induction of optimal antiviral humoral and protective response, as well as enhancement of B and T cell responses. We have shown previously that activation of the RIG-I pathway induces panantiviral effects both in vitro and in vivo (7,37). In the present study, we show that the activation of the RIG-I (RLR) pathway in the presence of an antigen offers dose-sparing effects and induced enhanced germi- The affinities of antibodies collected at 4 weeks following single immunization, as well as those collected at 6 weeks (2 weeks postbooster), were measured by calculating their avidity indices as described in Materials and Methods.…”

Section: Discussionmentioning

confidence: 99%

“…We have also shown that activation of the RIG-I pathway induces type I IFN and panantiviral effects both in vitro and in vivo (7,8). The role of cytokines, including type I IFN, in antiviral immunity is well known, and recent studies from our laboratory, as well others, highlight the way in which type I IFN can modulate adaptive immune responses (9)(10)(11)(12).…”

mentioning

confidence: 97%

Activation of the RIG-I Pathway during Influenza Vaccination Enhances the Germinal Center Reaction, Promotes T Follicular Helper Cell Induction, and Provides a Dose-Sparing Effect and Protective Immunity

Kulkarni

Rasheed

Bhaumik

et al. 2014

Self Cite

Pattern recognition receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type I interferons (IFNs). Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway for antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens coadministered with 5=ppp-double-stranded RNA (dsRNA), a RIG-I ligand, developed robust levels of hemagglutination-inhibiting antibodies, enhanced germinal center reaction, and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in the draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of the RIG-I pathway was able to reduce the antigen requirement by 10-to 100-fold in inducing optimal influenza-specific cellular and humoral responses, including protective immunity. The effects induced by 5=ppp-dsRNA were significantly dependent on type I IFN and IPS-1 (an adapter protein downstream of the RIG-I pathway) signaling but were independent of the MyD88-and TLR3-mediated pathways. Our results show that activation of the RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses, and this indicates the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines. IMPORTANCEThe recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate antiviral cytokines that help control viral replication and dissemination. In this study, we show that the RLR pathway can be effectively exploited to enhance adaptive immunity and protective immune memory against viral infection. Our results show that activation of the RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at low vaccine antigen doses. These results highlight the potential utility of exploiting the RIG-I pathway to enhance viralvaccine-specific immunity and have broader implications for designing better vaccines in general.

“…Human A549 (ATCC CCL-185) and chicken DF1 (ATCC CRL-12203) cells grown in 12-well cell culture plates were transfected with 1.0 g of plasmid DNA using TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI). As a positive control, cells were treated with in vitro-transcribed 5= triphosphate RNA (TP-RNA) (41). Flow cytometry for cell apoptosis.…”

Section: Methodsmentioning

confidence: 99%

“…We transfected A549 and DF1 cells with the PB1-F2 plasmids and measured IFN-␤ expression at various time points using the TaqMan assay qRT-PCR. We used an empty plasmid vector as a negative control and in vitro-transcribed 5= triphosphate RNA (TP-RNA) as a positive control for IFN-␤ induction (41). IFN-␤ expression levels were normalized to GAPDH and expressed as fold induction over empty-vector transfection.…”

Section: Evolution Of Pb1-f2 Truncations In Avian and Mammalian Virusesmentioning

confidence: 99%

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

Kamal

Kumar

Davis

et al. 2015

Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCEAlthough most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.