Influenza A virus hemagglutinin containing basolateral localization signal does not alter the apical budding of a recombinant influenza A virus in polarized MDCK cells.

Morphogenesis of influenza virus is a complex multistep process involving transport of all viral components as either individual or subviral components to the specified assembly site and interaction among the viral components in an ordered fashion to initiate the budding process. Envelope glycoprotein(s) is believed to be the major determinant in selecting the viral budding site since the majority of the viral glycoproteins are directed to the budding site independent of other viral components. Influenza viruses bud from the apical surface of polarized epithelial cells and all three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and M2, are also targeted independently to the apical surface. Since HA is the major viral envelope protein, we decided to test whether basolaterally expressed HA can make the virus bud from the basolateral surface. Accordingly, we introduced the tyrosine-based basolateral-sorting signal to the cytoplasmic tail of HA by changing Cys561 --> Tyr561 and generated a transfectant virus by reverse genetics. Compared to the parent WSN virus, the mutant virus (HAtyr virus) contained less HA on its envelope. While the wild-type (wt) HA was >95% apical, the mutated HA (HAtyr) was approximately 60% basolateral in both transfected and virus-infected polarized MDCK cells. Also, HAtyr protein exhibited a much higher rate of endocytosis than the wt HA, in both apical and basolateral surface of transfected as well as virus-infected cells. However, the HAtyr virus, similar to wt WSN virus, was seen to bud almost exclusively (>99%) from the apical side of polarized MDCK cells. This finding was confirmed by using neuraminidase to facilitate virus release, by treating the collected virus particles with trypsin to cleave HA0 --> HA1 and HA2, by protein analysis of released virus particles, and finally, by electron microscopy. Therefore HA, the major glycoprotein alone, does not determine the budding site, and other factor(s), possibly both viral and host, is responsible for selecting the budding site of influenza virus.