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

Ryota Sato,Naoki Takizawa,Takashi Okura,Madoka Kawahara,Fumitaka Momose,Yuko Morikawa

doi:10.3389/fmicb.2019.01857

Ryota Sato, Naoki Takizawa + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2019.01857

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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.

Highlights

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
Earlier studies with polarized MDCK cells have revealed that influenza A virus is assembled and buds at the apical plasma membrane (PM), whereas vesicular stomatitis virus buds at the basolateral PM, (Rodriguez-Boulan and Sabatini, 1978; Rodriguez-Boulan and Pendergast, 1980)
Influenza A virus HA has been widely used as a tool for studying apical protein transport, and a study, using an adenovirus vector, has shown that HA is transported through ASE/AEE but not ARE in fully polarized MDCK cells (Cresawn et al, 2007)

Summary

Introduction

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. They are prototypes for type I and II transmembrane proteins, respectively, and have long been used for studies on posttranslational modifications of protein and intracellular membrane trafficking. Influenza A virus binds to a sialic acid receptor via HA1 and is internalized to endosomes, where virus-cell membrane fusion involving HA2 occurs (Knossow et al, 1984; Gamblin and Skehel, 2010; Hamilton et al, 2012; Sriwilaijaroen and Suzuki, 2012). It is well known that HA forms a homotrimer, whereas NA forms a homotetramer

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