Date Published: March 6, 2014
Publisher: Public Library of Science
Author(s): Seema S. Lakdawala, Yicong Wu, Peter Wawrzusin, Juraj Kabat, Andrew J. Broadbent, Elaine W. Lamirande, Ervin Fodor, Nihal Altan-Bonnet, Hari Shroff, Kanta Subbarao, Mark T. Heise.
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 viruses cause severe annual morbidity and mortality . The genome of influenza A viruses is composed of 8 negative-sense single stranded RNA gene segments (PB2, PB1, PA, HA, NP, NA, M and NS) that encode 10 major proteins and several auxiliary peptides. Production of infectious progeny virions requires incorporation of all 8 vRNA segments and occurs at the apical membrane of infected cells . The segmented nature of the viral genome allows for the generation of novel reassortant viruses containing genes from distinct parental viruses in co-infected cells. The 2009 pandemic H1N1 virus arose from a reassortment event between two swine origin viruses . Additionally, the 2013 H7N9 virus responsible for the outbreak in Mainland China and Taiwan is a reassortant virus deriving gene segments from avian H9N2 and H7N9 viruses . Reassortment provides an evolutionary advantage for influenza viruses and thus reassortant viruses pose a major public health risk. Understanding how vRNA segments are assembled and packaged into progeny virions is key for unlocking how reassortant viruses are generated, yet little is known about this process.
Reassortment of influenza virus gene segments can lead to the emergence of novel influenza viruses with pandemic potential. The data from this study provide insight into vRNA assembly, which is important for understanding the intracellular mechanism of vRNA reassortment. First, our data suggest that vRNA segments are not exported as individual segments since the majority of foci at the external nuclear periphery contain more than one vRNA segment (Figure 2C). Second, we observed many cytoplasmic foci with fewer than 4 vRNA segments (Figure 1, 2A, 2C and Figure S3), implying that all 8 vRNA segments are not exported from the nucleus together. Third, individual vRNA segments do not reach the plasma membrane separately since many foci contain more than a single vRNA segment and foci can fuse together in the cytoplasm (Figure 1, 2B, and 6C). Therefore, we believe that vRNA assembly is an active process that includes the formation of flexible subcomplexes that export from the nucleus and then undergo further assembly en route to the plasma membrane via dynamic colocalization events (Figure 7).