The role of acidic residues in the ?fusion segment? of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Nobusawa, Eri; Hishida, Ryuichi; Murata, Masayuki; Kawasaki, Kazunori; Ohnishi, Shun‐ichi; Nakajima, Kazuki

doi:10.1007/bf01314963

Cited by 13 publications

(10 citation statements)

References 26 publications

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“…Therefore, the target size of the region responsible for fusion might be small. Furthermore, Nobusawa et al (17), using an HA protein expression system, found that several amino acid changes in the HA fusion domain did not affect fusion activity. Therefore, it is not likely that amino acid changes were more restricted on HA2 than on HA1.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Nakajima¹,

Nobusawa²,

Tonegawa

et al. 2003

J Virol

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We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.

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Section: Discussionmentioning

confidence: 99%

“…Fusion assay was performed as described previously (17). At 40 h posttransfection, the medium was removed.…”

mentioning

confidence: 99%

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Nakajima¹,

Nobusawa²,

Tonegawa

et al. 2003

J Virol

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“…The syncytium formation assay was performed as described previously with modifications (24). At 48 h posttransfection, HAexpressing Cos cells were incubated with 10 g of TPCK-trypsin/ml for 15 min at 37°C to cleave HA0 into HA1 and HA2.…”

Section: Cells and Virusmentioning

confidence: 99%

Influence of Acylation Sites of Influenza B Virus Hemagglutinin on Fusion Pore Formation and Dilation

Ujike

Nakajima

Nobusawa

2004

J Virol

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The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other singleacylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.Membrane fusion is an important event for enveloped viruses in the early stage of infection. Enveloped viruses inject their genomes into host cells by membrane fusion, which is mediated by particular viral proteins. Among these, hemagglutinin (HA) of influenza A virus (AHA) is the best-characterized viral protein and has been shown to induce low-pH-dependent membrane fusion (30). HA is a type I homotrimeric glycoprotein, and the precursor of each of its monomers is synthesized as a single polypeptide chain. After virus budding or during intracellular transport, the HA precursor is processed by proteolytic enzymes to generate two disulfide-linked subunits, HA1 and HA2. This processing creates a new amino terminus on HA2, referred to as the fusion peptide. In addition to fusion activity, HA is also responsible for binding of the virus to cell surface receptors (14). Since determination of the three-dimensional structure of H3 HA by X-ray analysis, the functional regions, i.e., the receptor-binding site and the fusion peptide, have been located on three-dimensional models (36).Following the entry of influenza viruses into host cells through receptor-mediated endocytosis, an acidic environment in the endosome triggers a conformational change in HA. Details of the conformational change were recently demonstrated by X-ray analysis of a fragment of low-pH-treated HA and by studies of recombinant, bacterially expressed HA2 (2, 3, 4). These ...

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“…To examine how the increase in the hydrophobicity of the TM and/or CT affected fusion pore dilation, we performed a syncytium formation assay which was carried out as described previously (18,23). In brief, HAexpressing cells were treated with TPCK-trypsin, incubated in acidic medium (pH 5.0) for 5 min at 37 C, and neutralized.…”

Section: Note (Facs)mentioning

confidence: 99%

Influence of Additional Acylation Site(s) of Influenza B Virus Hemagglutinin on Syncytium Formation

Ujike

Nakajima

Nobusawa

2005

Microbiology and Immunology

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Abstract:We studied the effects of an increase in the hydrophobicity of the transmembrane domain (TM) and cytoplasmic tail (CT) of influenza B virus hemagglutinin (BHA) on fusion activities. For this purpose, we created mutant HAs with novel acylation site(s) in the TM and/or CT. All mutants were able to induce hemifusion and to form fusion pores as well as could wild type (wt) BHA. However, the ability of these mutants to form syncytia was impaired, indicating that the increase in the hydrophobicity of these domains (especially the CT) affected fusion pore dilation.

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The role of acidic residues in the ?fusion segment? of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Cited by 13 publications

References 26 publications

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Influence of Acylation Sites of Influenza B Virus Hemagglutinin on Fusion Pore Formation and Dilation

Influence of Additional Acylation Site(s) of Influenza B Virus Hemagglutinin on Syncytium Formation

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