Abstract

Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and "just-in-time" universal vaccination against all IAV strains, including drug-resistant pandemics. One Sentence SummaryTargeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo.