Dataset Information

Quantitative Live Cell Imaging Reveals Differential Manipulation of Rab11A Transport Pathways by Influenza virus and RSV

ABSTRACT: Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/s, 330 nm isotropic spatial resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection. Here we report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with RSV alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport between Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Influenza A Virus (a/california/07/2009(h1n1)) Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Influenza

SUBMITTER: Katarzyna Kulej

LAB HEAD: Benjamin A. Garcia

PROVIDER: PXD016331 | Pride | 2020-01-14

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20190418_LakdawalaSS_RAB11A_IPeluate_A_CA0709_H1N1_Rep1_Fraction.mgf	Mgf
	20190418_LakdawalaSS_RAB11A_IPeluate_A_CA0709_H1N1_Rep1_Fraction.mzML	Mzml
	20190418_LakdawalaSS_RAB11A_IPeluate_A_CA0709_H1N1_Rep1_Fraction.mzid.gz	Mzid
	20190418_LakdawalaSS_RAB11A_IPeluate_A_CA0709_H1N1_Rep1_Fraction.pep.xml	Pepxml
	20190418_LakdawalaSS_RAB11A_IPeluate_A_CA0709_H1N1_Rep1_Fraction.pride.mgf.gz	Mgf

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Publications

Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association.

Bhagwat Amar R AR Le Sage Valerie V Nturibi Eric E Kulej Katarzyna K Jones Jennifer J Guo Min M Tae Kim Eui E Garcia Benjamin A BA Weitzman Matthew D MD Shroff Hari H Lakdawala Seema S SS

Nature communications 20200107 1

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increas ...[more]

PMID: 31911620

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Project description:Paramyxoviruses are negative sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) generates heterogenous populations of cells, with cells enriched in full-length virus producing viral particles containing standard or defective viral genomes, while cells enriched in DVGs did not, despite high levels of defective viral genome replication. Here we take advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examine the role of matrix protein and nucleoprotein and determine that they are not sufficient to drive interaction of vRNPs and recycling endosomes. Then, using a combination of mass spectrometry and comparative protein abundance and localization in DVG- and FL-high cells, we identify viral polymerase complex components L and, specifically, its cofactor C proteins as interactors with Rab11a. We find that accumulation of these proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells which remain in replication phases. Paramyxoviruses are a family of viruses that include a number of pathogens with significant burdens on human health. Particularly, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children and do not have any vaccines or direct acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and coopt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells, yet the viral proteins required for viral engagement with the recycling endosome pathway is not known. Here we use the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step in viral assembly. We find that viral polymerase components large protein L and accessory C proteins are necessary for engagement with recycling endosomes. These findings are important in identifying viral targets for the development of antivirals.