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Abstract
It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.
Highlights
The segmented nature of the influenza A virus (IAV) genome poses a challenge, as it necessitates selective and efficient packaging of 8 distinct genomic RNAs into a nascent virion to form an infectious virus particle
The influenza A virus (IAV) genome is composed of 8 distinct RNA segments
The current model for IAV genome packaging suggests that newly synthesized viral ribonucleoprotein complexes are exported out of the nucleus and trafficked across the cytoplasm to the plasma membrane for egress [2,3]. vRNPs are trafficked on vesicles that are positive for the host factor Rab11a using the microtubule or actin networks [4,5,6,7]
Summary
The segmented nature of the influenza A virus (IAV) genome poses a challenge, as it necessitates selective and efficient packaging of 8 distinct genomic RNAs into a nascent virion to form an infectious virus particle. It has been proposed that association of the 8 individual vRNPs occurs during transport across the cytoplasm; studies suggest that the process of vRNP association can begin in the nucleus [6,7,8,9]. It remains to be determined how 8 distinct vRNP complexes are brought together for specific genome assembly. Developing new strategies to fluorescently tag NP will allow us to investigate how cellular factors interact with vRNP complexes to facilitate the transport, assembly, and packaging of vRNPs into nascent virions
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