Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches.
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References
1.
TaubenbergerJK, ReidAH, JanczewskiTA and FanningTG. (2001). Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc Lond B Biol Sci, 356:1829–1839.
TaubenbergerJK and MorensDM. (2009). Pandemic influenza-including a risk assessment of H5N1. Rev Sci Tech, 28:187–202.
4.
ParrishCR, MurciaPR and HolmesEC. (2015). Influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans. J Virol, 89:2990–2994.
5.
DundonWG, HeidariA, FusaroA, MonneI, BeatoMS, CattoliG, KochG, StarickE, BrownIH, et al. (2012). Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011—review and recommendations for surveillance. Vet Microbiol, 154:209–221.
6.
SchwartzmanLM, CathcartAL, PujanauskiLM, QiL, KashJC and TaubenbergerJK. (2015). An intranasal virus-like particle vaccine broadly protects mice from multiple subtypes of influenza A virus. MBio, 6:e01044.
7.
FouchierRA, MunsterV, WallenstenA, BestebroerTM, HerfstS, SmithD, RimmelzwaanGF, OlsenB and OsterhausAD. (2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol, 79:2814–2822.
8.
CohenAL, HellfersceeO, PretoriusM, TreurnichtF, WalazaS, MadhiS, GroomeM, DawoodH, VariavaE, et al. (2014). Epidemiology of influenza virus types and subtypes in South Africa, 2009–2012. Emerg Infect Dis, 20:1162–1169.
9.
DawoodFS, JainS, FinelliL, ShawMW, LindstromS, GartenRJ, GubarevaLV, XuX, BridgesCB and UyekiTM. (2009). Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med, 360:2605–2615.
10.
UyekiTM and CoxNJ. (2013). Global concerns regarding novel influenza A (H7N9) virus infections. N Engl J Med, 368:1862–1864.
11.
YenHL and WebsterRG. (2009). Pandemic influenza as a current threat. Curr Top Microbiol Immunol, 333:3–24.
12.
BouvierNM and PaleseP. (2008). The biology of influenza viruses. Vaccine, 26:D49–D53.
13.
SamjiT. (2009). Influenza A: understanding the viral life cycle. Yale J Biol Med, 82:153–159.
14.
YamayoshiS, WatanabeM, GotoH and KawaokaY. (2016). Identification of a novel viral protein expressed from the PB2 segment of influenza A virus. J Virol, 90:444–456.
15.
BalgiAD, WangJ, ChengDYH, MaC, PfeiferTA, ShimizuY, AndersonHJ, PintoLH, LambRA, DeGardoWF and RobergeM. (2013). Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay. PLoS One, 8:e55271.
16.
HaydenFG. (2001). Newer influenza antivirals, biotherapeutics and combinations. Influenza Other Respir Viruses, 7:63–75.
17.
MosconaA. (2005). Drug therapy—neuraminidase inhibitors for influenza. N Engl J Med, 353:1363–1373.
18.
JeffersonT, DemicheliV, Di PietrantonjC and RivettiD. (2006). Amantadine and rimantadine for influenza A in adults. Cochrane Database Syst Rev, 19:CD001169.
19.
BrightRA, MedinaMJ, XuX, Perez-OronozG, WallisTR, DavisXM, PovinelliL, CoxNJ and KlimovAI. (2005). Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet, 366:1175–1181.
20.
HurtAC, HolienJK, ParkerMW and BarrIG. (2009). Oseltamivir resistance and the H274Y neuraminidase mutation in seasonal, pandemic and highly pathogenic influenza viruses. Drugs, 69:2523–2531.
21.
NeumannG, NodaT and KawaokaY. (2009). Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature, 459:931–939.
22.
PolandGA, JacobsonRM and OvsyannikovaIG (2009). Influenza virus resistance to antiviral agents: a plea for rational use. Clin Infect Dis, 48:1254–1256.
23.
WatanabeT, KawakamiE, ShoemakerJE, LopesTJ, MatsuokaY, TomitaY, Kozuka-HataH, GoraiT, KuwaharaT, et al. (2015). Influenza virus-host interactome screen as a platform for antiviral drug development. Cell Host Microbe, 16:795–805.
24.
RoehrB. (1998). Fomivirsen approved for CMV retinitis. J Int Assoc Physicians AIDS Care, 4:14–16.
25.
CrookeST and GearyRS. (2013). Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B. Br J Clin Pharmacol, 76:269–276.
26.
MathewsDH. (2005). Predicting a set of minimal free energy RNA secondary structures common to two sequences. Bioinformatics, 21:2246–2253.
27.
LenartowiczE, KesyJ, RuszkowskaA, Soszynska-JozwiakM, MichalakP, MossWN, TurnerDH, KierzekR and KierzekE. (2016). Self-folding of naked segment 8 genomic RNA of influenza A virus. PLoS One, 11:e0148281.
28.
KierzekE and KierzekR. (2003). The synthesis of oligoribonucleotides containing N-6-alkyladenosines and 2-methylthio-N-6-alkyladenosines via post-synthetic modification of precursor oligomers. Nucleic Acids Res, 31:4461–4471.
29.
XiaTB, SantaLuciaJ, BurkardME, KierzekR, SchroederSJ, JiaoXQ, CoxC and TurnerDH. (1998). Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry, 37: 14719–14735.
30.
MathewsDH. (2004). Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization. RNA, 10:1178–1190.
31.
BreenM, NogalesA, BakerSF, PerezDR, Martínez-SobridoL. (2016). Replication-competent influenza A and B viruses expressing a fluorescent dynamic timer protein for in vitro and in vivo studies. PLoS One, 11:e0147723.
32.
VijaykrishnaD, MukerjiR and SmithGJ. (2015). RNA Virus reassortment: an evolutionary mechanism for host jumps and immune evasion. PLoS Pathog, 11:e1004902.
33.
WatanabeT, ZhongG, RussellCA, NakajimaN, HattaM, HansonA, McBrideR, BurkeDF, TakahashiK, et al. (2015). Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential. Cell Host Microbe, 15:692–705.
34.
FraserC, DonnellyCA, CauchemezS, HanageWP, Van KerkhoveMD, HollingsworthTD, GriffinJ, BaggaleyRF, JenkinsHE, et al. (2009). Pandemic potential of a strain of influenza A (H1N1): early findings. Science, 324:1557–1561.
35.
InagakiA, GotoH, KakugawaS, OzawaM and KawaokaY. (2012). Competitive incorporation of homologous gene segments of influenza A virus into virions. J Virol, 86:10200–10202.
36.
HattaT, TakaiK, NakadaS, YokotaT and TakakuH. (1997). Specific inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: penetration and localization of oligonucleotides in clone 76 cells. Biochem Biophys Res Commun, 232:545–549.
37.
AbeT, MizutaT, HattaT, Miyano-KurosakiN, FujiwaraM, TakaiK, ShigetaS, YokotaT and TakakuH. (2001). Antisense therapy of influenza. Eur J Pharm Sci, 13:61–69.
38.
GeQ, PasteyM, KobasaD, PuthavathanaP, LupferC, BestwickRK, IversenPL, ChenJ and SteinDA. (2006). Inhibition of multiple subtypes of influenza A virus in cell cultures with morpholino oligomers. Antimicrob Agents Chemother, 50:3724–3733.
39.
GabrielG, NordmannA, SteinDA, IversenPL and KlenkHD. (2008). Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus. J Gen Virol, 89:939–948.
40.
LupferC, SteinDA, MourichDV, TepperSE, IversenPL and PasteyM. (2008). Inhibition of influenza A H3N8 virus infections in mice by morpholino oligomers. Arch Virol, 153:929–937.
41.
ZhangT, ZhaoPS, ZhangW, LiangM, GaoYW, YangST, WangTC, QinC, WangCY and XiaXZ. (2011). Antisense oligonucleotide inhibits avian influenza virus H5N1 replication by single chain antibody delivery system. Vaccine, 29:1558–1564.
42.
Soszynska-JozwiakM, MichalakP, MossWN, KierzekR and KierzekE. (2015). A conserved secondary structural element in the coding region of the influenza A virus nucleoprotein (NP) mRNA is important for the regulation of viral proliferation. PLoS One, 10:e0141132.
43.
GiannecchiniS, WiseHM, DigardP, ClausiV, Del PoggettoE, VescoL, PuzelliS, DonatelliI and AzziA. (2011). Packaging signals in the 5′-ends of influenza virus PA, PB1, and PB2 genes as potential targets to develop nucleic-acid based antiviral molecules. Antiviral Res, 92:64–72.
44.
WuY, ZhangG, LiY, JinY, DaleR, SunLQ and WangM. (2008). Inhibition of highly pathogenic avian H5N1 influenza virus replication by RNA oligonucleotides targeting NS1 gene. Biochem Biophys Res Commun, 365:369–374.
45.
DuanM, ZhouZ, LinRX, YangJ, XiaXZ and WangSQ. (2008). In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide. Antivir Ther, 13:109–114.
46.
GiannecchiniS, ClausiV, NosiD and AzziA. (2009). Oligonucleotides derived from the packaging signal at the 5′ end of the viral PB2 segment specifically inhibit influenza virus in vitro. Arch Virol, 154:821–832.
47.
FujiiK, OzawaM, Iwatsuki-HorimotoK, HorimotoT and KawaokaY. (2009). Incorporation of influenza A virus genome segments does not absolutely require wild-type sequences. J Gen Virol, 90:1734–1740.
48.
FujiiK, FujiiY, NodaT, MuramotoY, WatanabeT, TakadaA, GotoH, HorimotoT and KawaokaY. (2005). Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions. J Virol, 79:3766–3774.
49.
WangH, FengZ, ShuY, YuH, ZhouL, ZuR, HuaiY, DongJ, BaoC, et al. (2008). Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China. Lancet, 371:1427–1434.
50.
UngchusakK, AuewarakulP, DowellSF, KitphatiR, AuwanitW, PuthavathanaP, UiprasertkulM, BoonnakK, PittayawonganonC, et al. (2005). Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med, 352:333–340.
51.
FratczakA, KierzekR and KierzekE. (2009). LNA-modified primers drastically improve hybridization to target RNA and reverse transcription. Biochemistry, 48:514–516.
52.
MathewsDH, BurkardME, FreierSM, WyattJR and TurnerDH. (1999). Predicting oligonucleotide affinity to nucleic acid targets. RNA, 5:1458–1469.
53.
LuZJ and MathewsDH. (2008). OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics. Nucleic Acids Res, 36:W104–W108.
54.
ArranzR, ColomaR, Javier ChichonF, Javier ConesaJ, CarrascosaJL, ValpuestaJM, OrtinJ and Martin-BenitoJ. (2012). The structure of native influenza virion ribonucleoproteins. Science, 338:1634–1637.
55.
ColomaR, ValpuestaJM, ArranzR, CarrascosaJL, OrtinJ and Martin-BenitoJ. (2009). The structure of a biologically active influenza virus ribonucleoprotein complex. PLOS Pathogens, 5:e1000491
56.
BaudinF, BachC, CusackS and RuigrokRWH. (1994). Structure of influenza-virus RNP. I. Influenza-virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. EMBO J, 13:3158–3165.
57.
HerschlagD. (1991). Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc Natl Acad Sci USA, 88:6921–6925.
58.
RobertsRW and CrothersDM. (1991). Specificity and stringency in DNA triplex formation. Proc Natl Acad Sci USA, 88:9397–9401.
59.
QuattroneA, PapucciL, SchiavoneN, MiniE and CapaccioliS. (1994). Intracellular enhancement of intact antisense oligonucleotide steady-state levels by cationic lipids. Anticancer Drug Des, 9:549–553.
60.
HutchinsonEC, von KirchbachJC, GogJR and DigardP. (2010). Genome packaging in influenza A virus. J Gen Virol, 91:313–328.
61.
NodaT, SugitaY, AoyamaK, HiraseA, KawakamiE, MiyazawaA, SagaraH and KawaokaY. (2012). Three-dimensional analysis of ribonucleoprotein complexes in influenza A virus. Nat Commun, 3:639.
62.
FournierE, MoulesV, EssereB, PaillartJC, SirbatJD, IselC, CavalierA, RollandJP, ThomasD, LinaB and MarquetR. (2012). A supramolecular assembly formed by influenza A virus genomic RNA segments. Nucleic Acids Res, 40:2197–2209.
63.
FournierE, MoulesV, EssereB, PaillartJC, SirbatJD, CavalierA, RollandJP, ThomasD, LinaB, IselC and MarquetR. (2012). Interaction network linking the human H3N2 influenza A virus genomic RNA segments. Vaccine, 30:7359–7367.
64.
GavazziC, IselC, FournierE, MoulesV, CavalierA, ThomasD, LinaB and MarquetR. (2013). An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus. Nucleic Acids Res, 41:1241–1254.
65.
GavazziC, YverM, IselC, SmythRP, Rosa-CalatravaM, LinaB, MoulesV and MarquetR. (2013). A functional sequence-specific interaction between influenza A virus genomic RNA segments. Proc Natl Acad Sci USA, 110:16604–16609.
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