Abstract
Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.
Highlights
Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA
The virus contains an eight-partite RNA genome, with each segment encapsidated as an individual viral ribonucleoprotein complex. viral ribonucleoproteins (vRNPs) are composed of single-stranded negative-sense RNA, with base paired terminal sequences originating a double-stranded RNA portion to which binds the trimeric RNAdependent RNA polymerase (RdRp), composed of PB1, PB2, and PA
We assessed the formation of vRNP hotspots and the subcellular distribution of Rab[11] in a mini-replicon system expressing a single segment type, segment 7 of influenza A/Puerto Rico/34/8 (PR8)
Summary
We show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. Each puncta accommodates different vRNP segments with the diversity in vRNPs increasing with proximity to the plasma membrane[15] These observations led to the proposal that genome assembly precedes vRNP packaging into budding virions by a process linked with the formation of the referred vRNP hotspots[12,14,15,16]. We propose that viral inclusion formation precedes and facilitates stochastic vRNP–vRNP interactions in a liquid environment of crowded vRNPs
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