Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments

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.

Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes

“…To further corroborate the role of Rab11a in the transport of vRNPs across TNTs, we used Rab11a knockout [KO]A549 cells generated by CRISPR/Cas9 [ 12 ] and wild type [WT]A549 cells as a control. As before, cells were infected with either NL09 or P99 viruses and then stained for NP, Rab11a and F-Actin.…”

“…Further studies are needed to determine whether the same transport mechanism is active within TNTs and, conversely, whether Rab11a-actin dynamics may function in vRNP transport both within and between cells. Although Rab11a has been shown to be important for trafficking and efficient assembly of IAV genomes [ 2 , 8 , 12 ], we have shown that the loss of Rab11a does not completely abrogate genome assembly and viral particle release, indicating that there may exist an alternative pathway for canonical assembly of virions [ 12 ].…”

“…Generation and characterization of Rab11a KO A549 cells wasreported in [ 12 , 46 ]. Briefly, two guide RNAs [gRNA] targeting the promoter and exon 1 of the Rab11a gene were used.…”

“…There are, however, conflicting observations about the impact of microtubule disruptionon the viral lifecycle, with results ranging from no detectable effect [ 6 ] to an attenuation of viral progeny production [ 8 ]. Our prior work revealed that loss of Rab11a reduces infectious viral titers, most likely due to a defect in the packaging of vRNPs, leading to the formation of incomplete viral particles [ 12 ]. Taken together, these data demonstrate the importance of an intact microtubule network as well as Rab11a in the IAV life cycle.…”

The C-Terminal Domains of the PB2 Subunit of the Influenza A Virus RNA Polymerase Directly Interact with Cellular GTPase Rab11a

“…More recently, an alternative model has been proposed in which Rab11a and vRNPs are recruited to membranes of remodeled endoplasmic reticulum (ER), leading to the biogenesis of irregularly coated vesicles (ICVs), which ensure transport of vRNPs from the ER to the plasma membrane ( 22 ). Rab11a has also been implicated in the formation of liquid organelles promoting genome assembly and mediating cell-cell spread and reassortment of IAV genomes via tunneling nanotubes ( 23 – 25 ).…”