Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

Abstract

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

“…provide the molecular basis underlying a viral-induced mechanism to alter host recycling efficiency that promotes vesicular clustering and originates from the Rab11-dependent vRNP hotspots previously described (Chou et al, 2013;Lakdawala et al, 2014). We show that vRNP binding to Rab11, and no other viral component, induced alterations in Rab11 distribution (Figs 1A-C and 2), and that infection decreased recycling of transferrin (Fig.…”

“…Segmentation imposes a problem on viral assembly, because eight-segment vRNPs need to meet in order to form an infectious virion. Although supramolecular assembly of vRNP complexes could take place at the surface, several reports suggest that it occurs en route to the plasma membrane (Chou et al, 2013;Eisfeld et al, 2011;Lakdawala et al, 2014;Momose et al, 2011). In this paper, we Fig.…”

“…Given their function, it has been postulated that the recycling endosome transports vRNPs to the surface using the microtubule network (Amorim et al, 2011;Momose et al, 2011). Later, it was shown that several vRNPs colocalize in a Rab11-dependent manner in cytosolic puncta that increase in size with infection, which led to the proposal that assembly of the 8-segment core occurs en route to the surface (Chou et al, 2013;Lakdawala et al, 2014). Collectively, these data place Rab11a as being central for viral assembly, with many unresolved questions.…”

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

“…Interestingly, the HMPV strategy for replication and transcription involves the formation of a reduced number of IBs, which are always located in the cell cytoplasm, usually within a distance of 5 m from the nucleus. This strategy differs drastically from that of influenza and hepatitis C virus replication and transcription, which were shown to occur at multiple small replicative sites dispersed across the cell nuclei or cytoplasm (47,48). Nevertheless, the formation of IBs for replication seems to be a common strategy among NNS viruses.…”

“…To our knowledge, this is the first study to image HMPV genome replication and one of the very few to image the genome replication of paramyxo-or pneumoviruses. To achieve detection of HMPV genomic and antigenomic RNAs, a FISH strategy similar to that used for imaging the replication of influenza and hepatitis C viruses was used (47,48). The use of multiple labeled probes complementary to a viral RNA target theoretically increases sensitivity up to the level of single molecules.…”