Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

Abstract

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold-or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that (i) the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and (ii) the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

“…Sphingolipid-and cholesterol-containing rafts have been proposed to be important for apical transport in polarized epithelial cells (55). Wild-type HA, NA, and M 2 are expressed at the apical surface of polarized cells (19,24,49). Previous studies indicated that alteration of residues in the TM domains of HA and NA can affect both DIG association and apical targeting, although the correlation was not absolute (28, 34, 53).…”

Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes

“…Since further deletion in the 3Ј end leaving only 15 nucleotides and of the 5Ј end leaving 268 nucleotides did not affect the efficiency of HA vRNA incorporation [compare HA(468)GFP(513) with HA(15)GFP(268)], we made additional deletion constructs by using pPolIHA (15)GFP(268), which possessed 15 nucleotides of the 3Ј end of the HA coding region and 268 nucleotides of the 5Ј end. Although the extent of vRNA incorporation was reduced gradually as the extent of deletions increased, 80 nucleotides in the 5Ј HA coding region seemed minimally required for efficient virion incorporation of the HA vRNA [compare HA (15) GFP(80) with HA(15)GFP (75)]. Further deletion analysis demonstrated that HA(9)GFP(80) with 9 nucleotides at the 3Ј end of the HA coding region resulted in efficient virion incorporation of the HA vRNA (Ͼ65%); however, the level of HA(9)GFP(80) vRNA present in transfected cells did not differ appreciably from that of HA(0)GFP(0) vRNA ( Fig.…”

The Sendai Virus Matrix Protein Appears to be Recruited in the Cytoplasm by the Viral Nucleocapsid to function in Viral Assembly and Budding

“…As the information for protein sorting is contained within the glycoprotein structure (Roth et aI., 1983;Jones et at., 1985;Stephens et al, 1986;Puddington et al, 1987), polarized budding can be viewed simply as the result of transport of M protein and nucleocapsids orchestrated by the glycoprotein.…”