Sequence of RNA segment 7 of the influenza B virus genome: Partial amino acid homology between the membrane proteins (M1) of Influenza A and B viruses and conservation of a second open reading frame

Briedis, Dalius J.; Lamb, Robert A.; Choppin, Purnell W.

doi:10.1016/0042-6822(82)90150-7

Cited by 86 publications

(31 citation statements)

References 16 publications

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“…However, influenza A viruses encode the M2 polypeptide that is translated from a spliced mRNA derived from RNA segment 7 and overlaps the region encoding M1 (24,27). No counterpart for M2 has been reported in influenza B virus-infected cells, although RNA segment 7 of influenza B virus, which encodes the Ml protein, does contain a second overlapping reading frame (4). It has recently been shown that influenza A virus M2 protein is an integral membrane protein expressed at the infected-cell surface (29,56 sizes (100 and 97 amino acids, respectively) and contain a single internal hydrophobic domain.…”

Section: Resultsmentioning

confidence: 99%

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

Williams¹,

Lamb²

1986

Mol. Cell. Biol.

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The membrane orientation of the NB protein of influenza B virus, a small (Mr,,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.In eucaryotic cells, glycoproteins are either translocated completely across the membrane into the lumen of the endoplasmic reticulum for soluble proteins destined for secretion or they are asymmetrically integrated into distinct cellular membranes. Integral membrane proteins may span the membrane once or more with the NH2 and COOH termini of the polypeptide chain on either side of the bilayer. These proteins are anchored in membranes by a membranespanning domain which is generally found to consist of 20 or more hydrophobic or uncharged amino acids. It has been established that the orientation of an integral membrane protein is absolute and is maintained regardless of the final destination of the protein (e.g., in the plasma membrane) (reviewed in references 3 and 54). Similarly, the addition of asparagine N-linked carbohydrate to an integral membrane protein is also asymmetric and occurs in the lumen of endoplasmic reticulum vesicles at the amino acid sequence Asn-X-Ser/Thr (reviewed in reference 17). However, determination of the transmembrane orientation and location of the precise sites used for addition of carbohydrate by peptide analysis are often difficult.We have been interested in examining properties of a glycoprotein, NB, of influenza B virus which circumstantial evidence indicated was an integral membrane protein. In the course of these studies we developed a method, based on site-specific mutagenesis and expression of mutant cDNAs in eucaryotic cells, that enabled us to determine the orientation of the protein in membranes and the precise sites of carbohydrate addition.Influenza B virus NB glycoprotein (100 amino acids) is translated from a bicistronic mRNA derived from RNA segment 6 that also encodes the neuraminidase (NA) glycoprotein by using overlapping reading frames (28,48). The first AUG codon in the mRNA is used for the initiation of NB and the second AUG codon, separated from the first AUG codon by four nucleotides, is used to initiate NA. NB * Corresponding author.

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Section: Resultsmentioning

confidence: 99%

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

Williams¹,

Lamb²

1986

Mol. Cell. Biol.

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“…One of the differences is found in RNA segment 7 of influenza B virus, which encodes the BM2 protein in addition to encoding M1 by bicistronic mRNA (4,12). The translational strategy of BM2 is quite unique.…”

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confidence: 99%

Influenza B Virus BM2 Protein Is a Crucial Component for Incorporation of Viral Ribonucleoprotein Complex into Virions during Virus Assembly

Imai

Watanabe

Ninomiya

et al. 2004

J Virol

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Influenza B virus contains four integral membrane proteins in its envelope. Of these, BM2 has recently been found to have ion channel activity and is considered to be a functional counterpart to influenza A virus M2, but the role of BM2 in the life cycle of influenza B virus remains unclear. In an effort to explore its function, a number of BM2 mutant viruses were generated by using a reverse genetics technique. The BM2⌬ATG mutant virus synthesized BM2 at markedly lower levels but exhibited similar growth to wild-type (wt) virus. In contrast, the BM2 knockout virus, which did not produce BM2, did not grow substantially but was able to grow normally when BM2 was supplemented in trans by host cells expressing BM2. These results indicate that BM2 is a required component for the production of infectious viruses. In the one-step growth cycle, the BM2 knockout virus produced progeny viruses lacking viral ribonucleoprotein complex (vRNP). The inhibited incorporation of vRNP was regained by trans-supplementation of BM2. An immunofluorescence study of virus-infected cells revealed that distribution of hemagglutinin, nucleoprotein, and matrix (M1) protein of the BM2 knockout virus at the apical membrane did not differ from that of wt virus, whereas the sucrose gradient flotation assay revealed that the membrane association of M1 was greatly affected in the absence of BM2, resulting in a decrease of vRNP in membrane fractions. These results strongly suggest that BM2 functions to capture the M1-vRNP complex at the virion budding site during virus assembly.Influenza A, B, and C viruses are enveloped viruses that assemble at the plasma membrane and bud from infected cells. Both influenza A and B viruses have eight single-stranded negative-sense RNA segments encapsidated with polymerase proteins and nucleoprotein (NP), forming a viral ribonucleoprotein complex (vRNP). The envelope contains three different membrane proteins, hemagglutinin (HA), neuraminidase (NA), and ion channel protein M2 in influenza A virus and NB in influenza B virus (20). The small proteins, M2 and NB, are transmembrane proteins which adopt an N-out C-in orientation (21,35,38) and are incorporated into virions as a relatively minor component (3, 37). On the other hand, in the case of influenza A virus, the most abundant product matrix (M1) protein has multiple functions and plays central roles in virus assembly. M1 interacts with vRNP in the nucleus to mediate nuclear export and to prevent nuclear reentry of vRNP (22,34) and with the cytoplasmic tail of HA and NA on the plasma membrane to form the inner surface of the lipid bilayer of the envelope (1,7,18,40). These interactions are thought to be essential for particle formation and virus release from infected cells.Although both influenza A and B viruses possess similarities in the coding strategy of genome RNA segments and in the function of viral proteins, there are some differences between these viruses. One of the differences is found in RNA segment 7 of influenza B virus, which encodes the BM2 prote...

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“…The BM2 protein is encoded by bicistronic mRNA derived from RNA segment 7 of influenza B virus, which also encodes the matrix protein M1 (5,13). The translational strategy of BM2 is unique.…”

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confidence: 99%

Influenza B Virus BM2 Protein Is Transported through the trans- Golgi Network as an Integral Membrane Protein

Watanabe

Imai

Ohara

et al. 2003

J Virol

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A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.

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Sequence of RNA segment 7 of the influenza B virus genome: Partial amino acid homology between the membrane proteins (M1) of Influenza A and B viruses and conservation of a second open reading frame

Cited by 86 publications

References 16 publications

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

Influenza B Virus BM2 Protein Is a Crucial Component for Incorporation of Viral Ribonucleoprotein Complex into Virions during Virus Assembly

Influenza B Virus BM2 Protein Is Transported through the trans- Golgi Network as an Integral Membrane Protein

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