Influenza A virus-generated small RNAs regulate the switch from transcription to replication

Abstract

The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22-27 nt in length and correspond to the 5' end of each of the viral genomic RNA (vRNA) segments. Expression of svRNA correlates with the accumulation of vRNA and a bias in RNA-dependent RNA polymerase (RdRp) activity from transcription toward genome replication. Synthesis of svRNA requires the RdRp, nucleoprotein and the nuclear export protein NS2. In addition, svRNA is detectable during replication of various influenza A virus subtypes across multiple host species and associates physically with the RdRp. We demonstrate that depletion of svRNA has a minimal impact on mRNA and complementary vRNA (cRNA) but results in a dramatic loss of vRNA in a segment-specific manner. We propose that svRNA triggers the viral switch from transcription to replication through interactions with the viral polymerase machinery. Taken together, the discovery of svRNA redefines the mechanistic switch of influenza virus transcription/replication and provides a potential target for broad-range, anti-influenza virus-based therapeutics.

Fig. 1.

Identification of influenza A virus-derived small RNAs. A549 cells were mock treated or infected with influenza A/PR/8/34 H1N1 virus at an MOI of 1. Twelve hours postinfection, total RNA was resolved on an SDS/PAGE gel, and RNA <40 nt in length was isolated and sequenced using SOLiD-based technology. Each of the eight segments (and corresponding accession numbers) and their ORFs are shown. Above each cartoon is a histogram depicting peaks of total reads captured per segment (labeled as n = total reads).

Fig. 2.

Influenza A virus-specific expression of svRNA. (A) Northern blot analysis of A549 cells mock treated or infected with influenza A/PR/8/34 virus at an MOI of 1. Total RNA was harvested at 4, 8, 12, 24, and 36 hpi. Extracts were resolved by denaturing gel electrophoresis and hybridized with a radiolabeled pan-specific svRNA probe. (B) Western blot analysis from duplicate samples as described in A. (C) qPCR analysis of NS genomic RNA from samples processed in A. Values presented are normalized to tubulin for each sample and are represented as fold induction over the mock-transfected sample. Error bars reflect SD of fold change. (D) Northern blot analysis of mock-, A/PR/8/34-, VSV-, or IFN−I–treated A549 cells. (E) Northern blot analysis of isolated allantoic membrane from embryonated chicken eggs mock treated or infected with A/PR/8/34 (H1N1), A/Panama/2007/99 (H3N2), or A/Vietnam/1203/04 (H5N1). (F) Northern blot analysis of human (HEK293), MDCK, and murine embryonic (MEF) fibroblasts mock treated or infected with A/PR/8/34 (H1N1). U6 was used as a loading control for all Northern blots.

Fig. 3.

In vitro generation and molecular interactions of svRNA. (A) Northern blot analysis of fibroblasts mock transfected, transfected with all eight bidirectional influenza A virus-encoding plasmids, or transfected with only seven of the eight bidirectional plasmids. Numbers above each lane indicate the missing segment. Total RNA was harvested 24 h posttransfection, resolved by denaturing gel electrophoresis, and hybridized with a radiolabeled pan-specific svRNA probe. (B) The upper two frames show Northern blots as in A with additional expression of either NEP/NS2 or NS1. The lower four frames show Western blots of total protein extract depicted in the upper two frames. (C) Northern blot analysis of svRNA from immunoprecipitated Flag-tagged proteins cotransfected with an svRNA pNS. (D) Northern blot analysis of svRNA mock treated or incubated with calf intestinal phosphatase (CIP). U6 was used as a loading control for all Northern blots.

Fig. 4.

Inhibition of svRNA suppresses vRNA synthesis. (A) Primer extension analysis of fibroblasts transfected with Scbl LNA or anti-HA and subsequently infected with A/PR8/34. 5S rRNA was used as a loading control. (B) Western blot analysis of HA, NP, NS1, and β-Actin from fibroblasts mock transfected or transfected with Scbl LNA or anti-HA and subsequently infected with influenza A/PR/8/34 for the indicated times. (C) Western blot analysis of HA, NP, NS1, and β-Actin for MDCKs mock infected or infected for 24 h with the indicated supernatants from B.

Fig. 5.

Anti-svRNA inhibits viral replication in a segment-specific manner. (A) Primer extension analysis of fibroblasts transfected with Scbl LNA or anti-HA,-NS, or -NA and subsequently infected with A/PR8/34. 5S rRNA was used as a loading control. (B) Western blot of HA, NP, and β-Actin for fibroblasts mock transfected or transfected with Scbl LNA or anti-HA LNA and subsequently infected with influenza A/PR/8/34 for the indicated times. (C) Viral titers for supernatants harvested at the indicated times for samples in B.