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Abstract

Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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/content/journal/acmi/10.1099/acmi.ac2019.po0145
2019-04-08
2024-03-28
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