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.
Highlights
The Influenza A virus genome consists of eight negative-sense, single-stranded RNAs
The viruses possess a genome consists of eight different RNA segments and the incorporation of all the eight RNA segments is required for the generation of an infectious virus particle
The precise process of how these eight viral RNA segments are co-packaged into progeny virus particles remains undefined due to the limitations of methodology to determine the locations of different vRNA segments in infected cells with single-molecule resolution
Summary
The Influenza A virus genome consists of eight negative-sense, single-stranded RNAs. In a virus particle or an infected cell, the viral RNAs exist in the form of viral ribonucleoprotein complexes (vRNPs) with the viral RNA (vRNA) encapsidated by the nucleoproteins (NP) and associate with the polymerase complex [1]. Nuclear localization signals on the NP protein are recognized by importin a (karyopherin a) and together the vRNP and importin a form a tri-complex with importin-b that is actively transported into the nucleus through the nuclear pore complex [7]. It has been shown with biochemical analysis that the replicating vRNPs were associated with densely packed chromatin while the newly synthesized vRNPs are released into the nucleoplasm [8]. The newly assembled vRNPs are exported into the cytoplasm, where
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