Apical Trafficking Pathways of Influenza A Virus HA and NA via Rab17- and Rab23-Positive Compartments

Abstract

The envelope proteins of influenza A virus, hemagglutinin (HA) and neuraminidase (NA), play critical roles in viral entry to host cells and release from the cells, respectively. After protein synthesis, they are transported from the trans -Golgi network (TGN) to the apical plasma membrane (PM) and assembled into virus particles. However, the post-TGN transport pathways of HA and NA have not been clarified. Temporal study by confocal microscopy revealed that HA and NA colocalized soon after their synthesis, and relocated together from the TGN to the upper side of the cell. Using the Rab family protein, we investigated the post-TGN transport pathways of HA and NA. HA partially colocalized with AcGFP-Rab15, Rab17, and Rab23, but rarely with AcGFP-Rab11. When analyzed in cells stably expressing AcGFP-Rab, HA/NA colocalized with Rab15 and Rab17, markers of apical sorting and recycling endosomes, and later colocalized with Rab23, which distributes to the apical PM and endocytic vesicles. Overexpression of the dominant-negative (DN) mutants of Rab15 and Rab17, but not Rab23, significantly delayed HA transport to the PM. However, Rab23DN impaired cell surface expression of HA. Live-cell imaging revealed that NA moved rapidly with Rab17 but not with Rab15. NA also moved with Rab23 in the cytoplasm, but this motion was confined at the upper side of the cell. A fraction of HA was localized to Rab17 and Rab23 double-positive vesicles in the cytoplasm. Coimmunoprecipitation indicated that HA was associated with Rab17 and Rab23 in lipid raft fractions. When cholesterol was depleted by methyl-β-cyclodextrin treatment, the motion of NA and Rab17 signals ceased. These results suggest that HA and NA are incorporated into lipid raft microdomains and are cotransported to the PM by Rab17-positive and followed by Rab23-positive vesicles.

Live Visualization of Hemagglutinin Dynamics during Infection by Using a Novel Reporter Influenza A Virus

“…Several researchers have engineered Influenza A viruses through reverse genetics to study different aspects of viral replication. These include insertion of Fluorescent or Bioluminescent reporters in to IAV structural proteins or expression of tetra cysteine or Sortase tags that allow chemical labelling of the viral protein for fluorescence-based detection [ 12 , 14 , 20 , 23 , 25 , 26 , 27 , 28 ]. Here, we focused on developing a reporter virus to achieve live imaging of HA dynamics in infected cells.…”

“…This assumption is supported by live imaging data, which showed reduced HA accumulation and translocation in presence of Tunicamycin. It has been reported that in polarized cells, HA transport occurs towards apical membrane [ 28 ]. In the live imaging experiment, we were able to observe lateral accumulation of HA at early time points, followed by apical transport at the late stages of infection.…”

The ESCRT-III molecules regulate the apical targeting of bile salt export pump

“…In contrast to sparse information on ESCRTs functioning on polarized sorting, a significant body of data suggested apical and basolateral membrane proteins traffic through Rab-associated endosomal compartments to target the destinated membrane domain in polarized cells [29,[46][47][48][49][50]. Studies in rodents [8,29] and polarized WIF-B9 cell models [10,33] have demonstrated the apical targeting of BSEP traversed Rab11-positive endosomal compartments.…”

Host–Virus Interaction: How Host Cells Defend against Influenza A Virus Infection

“…These proteins then assemble with viral genomic segments. The virions are finally closed and M1 and M2 proteins mediate virion budding from the apical side of the cells [28,[95][96][97][98]. NA protein cleavages the SA residues, which allows the virions to be released from the plasma membrane [99].…”