Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses (original) (raw)
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Journal of Virology, 2000
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 w...
Journal of Virology, 2000
Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.
Journal of Virology, 2004
Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant vir...
Journal of virology, 1992
The M2 protein of influenza A virus is a small, nonglycosylated transmembrane protein that is expressed on surfaces of virus-infected cells. A monoclonal antibody specific for the M2 protein was used to investigate its expression in polarized epithelial cells infected with influenza virus or a recombinant vaccinia virus that expresses M2. The expression of M2 on the surfaces of influenza virus-infected cells was found to be restricted to the apical surface, closely paralleling that of the influenza virus hemagglutinin (HA). Membrane domain-specific immunoprecipitation indicated that the M2 protein was inserted directly into the apical membrane with transport kinetics similar to those of HA. In polarized cells infected with a recombinant vaccinia virus that expresses M2, we found that 86 to 93% of surface M2 was restricted to the apical domain compared with 88 to 90% of HA in a similar assay. These results indicate that the M2 protein undergoes directional transport in the absence of...
Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins
Journal of Biological Chemistry, 2010
Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface glycoproteins of influenza A virus with their common ligand/ substrate: carbohydrate chains terminating in sialic acid. The specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid substrate of the virus neuraminidase enzyme but utilize the much tighter binding of the drugs for efficacy. Resistance to one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence of structural differences in the enzyme active site between the two phylogenetic groups. * This is the first article in the Thematic Minireview Series on Influenza Virus.
Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes
Biophysical Journal, 2010
The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membranerafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011
Linker and transmembrane region MALDI-TOF MS Phylogenetic groups of influenza virus Molecular modeling Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1-H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/ or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.
Membranes
Influenza A virus envelope contains lipid molecules of the host cell and three integral viral proteins: major hemagglutinin, neuraminidase, and minor M2 protein. Membrane-associated M1 matrix protein is thought to interact with the lipid bilayer and cytoplasmic domains of integral viral proteins to form infectious virus progeny. We used small-angle X-ray scattering (SAXS) and complementary techniques to analyze the interactions of different components of the viral envelope with M1 matrix protein. Small unilamellar liposomes composed of various mixtures of synthetic or “native” lipids extracted from Influenza A/Puerto Rico/8/34 (H1N1) virions as well as proteoliposomes built from the viral lipids and anchored peptides of integral viral proteins (mainly, hemagglutinin) were incubated with isolated M1 and measured using SAXS. The results imply that M1 interaction with phosphatidylserine leads to condensation of the lipid in the protein-contacting monolayer, thus resulting in formation ...