Colocalization of Different Influenza Viral RNA Segments in the Cytoplasm before Viral Budding as Shown by Single-molecule Sensitivity FISH Analysis

Abstract

The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

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.…”

“…In fact, Rab11 distribution changes with infection from discrete puncta to enlarged structures (Amorim et al, 2011;Chou et al, 2013;Eisfeld et al, 2011). This conclusion originated from confocal microscopy analyses from four independent studies, using different systems, including a mini-replicon system that was devoid of segments 4 and 6 (Amorim et al, 2011), and infection with wild-type viruses (Avilov et al, 2012a;Eisfeld et al, 2011;Momose et al, 2011).…”

“…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.…”

Early host response and immune signaling to 2009 pandemic influenza A (H1N1) viruses in primary cell culture models.

“…Briefly, importin protein importin-alpha recognizes the nuclear localization signals on IAV NP [130,131] , PB2 [132,133], or PA/PB1 [134,135], which is then recognized and bound to importin-beta transport receptor [136]. This complex diffuses through the nuclear pore complex and undergoes active RAN GTPase (RAN-GTP)-induced dissociation with the help of cytoplasmic RAN G-proteins, releasing the vRNP into the nucleus where transcription and replication take place [137,138].…”