Analysis of the Transmembrane Domain of Influenza Virus Neuraminidase, a Type II Transmembrane Glycoprotein, for Apical Sorting and Raft Association

Abstract

Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508-6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH 2 terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH 2 terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

“…Raft association appears necessary, but not sufficient, for apical sorting of hemaglutinin (4,49,80). In contrast, raft association is not absolutely required for apical sorting of neuraminidase (4,80).…”

“…Certain apical integral glycoproteins, including influenza viral proteins hemaglutinin and neuraminidase, associate with lipid rafts via their transmembrane domains (4,80). Although transmembrane domains of these proteins are important for both raft association and apical sorting, the signals for raft association and apical sorting are not identical (4,49,80).…”

“…Although transmembrane domains of these proteins are important for both raft association and apical sorting, the signals for raft association and apical sorting are not identical (4,49,80). Raft association appears necessary, but not sufficient, for apical sorting of hemaglutinin (4,49,80).…”

Role of Membrane Rafts in Viral Infection

“…HA, NA, NP and M2 independently utilize membrane rafts together with apical targeting signal sequence for the apical sorting process, leading to efficient preferential budding and release of progeny viruses from the apical surface membrane. However, direct interactions with membrane rafts are not necessarily essential for apical sorting of these viral proteins, indicating that they can also interact with apical sorting machineries outside their membrane rafts [150,[152][153][154]156]. For example, cellular protein VIP17/MAL, a raftassociated protein, is involved in apical transport of HA in dog kidney MDCK cells [192].…”

“…The role of membrane rafts in the assembly and budding of enveloped viruses has been investigated for influenza virus [59,63,[150][151][152][153][154][155][156][157][158][159][160], HIV-1 [63,[161][162][163][164][165][166][167][168][169][170][171][172][173], HTLV-1 [174], measles virus (Paramyxoviridae) [63,175,176], Sendai virus [Paramyxoviridae] [177,178], Newcastle disease virus (NDV; Paramyxoviridae) [179,180], RSV [147,148,[181][182][183][184], HSV [185,186], murine cytomegalovirus (MCMV; Herpesviridae) [187], Ebola virus [70,188], Marburg virus [70], and vesicular stomatitis virus (VSV; Rhabdoviridae) …”