TY - JOUR
T1 - Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
AU - Williams, Graham D.
AU - Townsend, Dana
AU - Wylie, Kristine M.
AU - Kim, Preston J.
AU - Amarasinghe, Gaya K.
AU - Kutluay, Sebla B.
AU - Boon, Adrianus C.M.
N1 - Funding Information:
NIGMS training grant 5T32GM007067 and NIAID ID Training Grant 2T32AI007172, and the Victoria Fraser Infectious Disease Research Fellowship provided funding to GW. This study was funded in part by the National Institute of Health (NIH) grants P01AI120943 (G.K.A) and R01-AI118938 (A.C.M.B.).
Funding Information:
We thank the following colleagues for comments on experiments comprising this manuscript: Michael Diamond, MD, David Wang, PhD, Daisy W. Leung, PhD, and Deborah Lenschow, MD and Anshu Gounder all of Washington University in Saint Louis. Additionally, Traci Bricker and Christopher Edwards provided technical support. Chris Brooke provided anti-M1 hybridoma, M2-1C6. NIGMS training grant 5T32GM007067 and NIAID ID Training Grant 2T32AI007172, and the Victoria Fraser Infectious Disease Research Fellowship provided funding to GW. This study was funded in part by the National Institute of Health (NIH) grants P01AI120943 (G.K.A) and R01-AI118938 (A.C.M.B.).
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.
AB - Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.
UR - http://www.scopus.com/inward/record.url?scp=85041285765&partnerID=8YFLogxK
U2 - 10.1038/s41467-018-02886-w
DO - 10.1038/s41467-018-02886-w
M3 - Article
C2 - 29386621
AN - SCOPUS:85041285765
SN - 2041-1723
VL - 9
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 465
ER -