Influenza A Virus Expresses High Levels of an Unusual Class of Small Viral Leader RNAs in Infected Cells

ABSTRACT Evidence has recently accumulated suggesting that small noncoding RNAs, and particularly microRNAs, have the potential to strongly affect the replication and pathogenic potential of a range of human virus species. Here, we report the use of deep sequencing to comprehensively analyze small viral RNAs (18 to 27 nucleotides [nt]) produced during infection by influenza A virus. Although influenza A virus differs from most other RNA viruses in that it replicates its genome in the nucleus and is therefore exposed to the nuclear microRNA processing factors Drosha and DGCR8, we did not observe any microRNAs encoded by influenza virus genes. However, influenza virus infection did induce the expression of very high levels—over 100,000 copies per cell by 8 h postinfection—of a population of 18- to 27-nt small viral leader RNAs (leRNAs) that originated from the precise 5′ ends of all eight influenza virus genomic RNA (vRNA) segments. Like the vRNAs themselves, our data indicate that the leRNAs also bear a 5′-terminal triphosphate and are therefore not capable of functioning as microRNAs. Instead, the high-level production of leRNAs may imply a role in another aspect of the viral life cycle, such as regulation of the switch from viral mRNA transcription to genomic RNA synthesis. IMPORTANCE Influenza A virus is an important human pathogen that has the potential to give rise to serious pandemics. Here, we demonstrate that influenza A virus induces the expression of very high levels of small viral leader RNAs (leRNAs) within hours of infection. These RNAs are unusual in that they bear a 5′ triphosphate and originate from the very 5′ ends of the eight viral genomic RNA (vRNA) segments. Their high expression may imply an important role in the viral life cycle that could potentially serve as a novel target for antiviral drugs.