scholarly journals Recognition of influenza A matrix protein by HLA-A2-restricted cytotoxic T lymphocytes. Use of analogues to orientate the matrix peptide in the HLA-A2 binding site.

1988 ◽  
Vol 168 (6) ◽  
pp. 2045-2057 ◽  
Author(s):  
F Gotch ◽  
A McMichael ◽  
J Rothbard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Influenza A ◽  
Matrix Protein ◽  
Single Amino Acid ◽  
The Matrix ◽  
Natural Peptide

CTL specific for the influenza A virus matrix peptide 57-68 and restricted by HLA-A2 were studied. Their ability to recognize a set of analogue peptides, each of which differed from the natural peptide by a single amino acid, was analyzed. This revealed a core of five amino acids, 61-65, where one or more changes completely abrogated recognition. The glycine at position 61 was the only residue where no substitution was tolerated. Analogue peptides that did not induce CTL-mediated lysis were tested as competitors with the natural peptide; those with substitutions at positions 60, 64, and 65 inhibited, identifying residues that interact with the TCR. Another approach was to test a set of four CTL clones on all of the analogues. Marked differences in recognition by individual CTL clones were observed for several substituted peptides. The data indicate that most of the analogues bind to HLA-A2 with possible differences in fine positioning of the peptide. An alpha helical orientation for the peptide is discussed.

scholarly journals HLA B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes.

1986 ◽  
Vol 164 (5) ◽  
pp. 1397-1406 ◽  
Author(s):  
A J McMichael ◽  
F M Gotch ◽  
J Rothbard
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Synthetic Peptide ◽  
Influenza A ◽  
Cytotoxic T Cells ◽  
Human Influenza ◽  
Infected Cells

Human influenza A virus-specific, cytotoxic T cells have been shown previously to recognize the virus nucleoprotein on infected cells. CTL preparations from four HLA B37-positive donors were shown to recognize a synthetic peptide that corresponded to amino acids 335-349 of the nucleoprotein sequence. Influenza-specific CTL from 10 donors of other HLA types failed to recognize this epitope. CD8+ CTL lines were derived from lymphocytes of two HLA B37-positive donors and used to show that the peptide was represented on virus-infected cells and to determine the probable boundaries of the epitope.

Recognition by HLA-A2-restricted cytotoxic T lymphocytes of endogenously generated and exogenously provided synthetic peptide analogues of the influenza a virus matrix protein

1993 ◽  
Vol 37 (4) ◽  
pp. 252-258 ◽  
Author(s):  
Samir Y. Sauma ◽  
Maureen C. Gammon ◽  
Maria A. Bednarek ◽  
Barry Cunningham ◽  
William E. Biddison ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Synthetic Peptide ◽  
Influenza A ◽  
Matrix Protein ◽  
Peptide Analogues

scholarly journals Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase

2010 ◽  
Vol 55 (2) ◽  
pp. 696-702 ◽  
Author(s):  
Kerstin Wunderlich ◽  
Mindaugas Juozapaitis ◽  
Charlene Ranadheera ◽  
Ulrich Kessler ◽  
Arnold Martin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Affinity ◽  
Influenza A Virus ◽  
Influenza A ◽  
Single Amino Acid ◽  
Binding Region ◽  
Binding Studies ◽  
Relationship Analysis ◽  
The Core

ABSTRACTThe influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

scholarly journals Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

2005 ◽  
Vol 79 (17) ◽  
pp. 11239-11246 ◽  
Author(s):  
E. G. M. Berkhoff ◽  
E. de Wit ◽  
M. M. Geelhoed-Mieras ◽  
A. C. M. Boon ◽  
J. Symons ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Influenza A ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Functional Constraints ◽  
Ctl Epitopes

ABSTRACT Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M158-66. We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M158-66 epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.

Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA-A2 in its recognition by cytotoxic T lymphocytes

1992 ◽  
Vol 22 (4) ◽  
pp. 903-907 ◽  
Author(s):  
Joanne Morrison ◽  
John Elvin ◽  
France Latron ◽  
Frances Gotch ◽  
Robert Moots ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Influenza A ◽  
Matrix Protein ◽  
Nonamer Peptide

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
Author(s):  
Daniel R. Perez ◽  
Ruben O. Donis
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

scholarly journals Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Nancy Hom ◽  
Lauren Gentles ◽  
Jesse D. Bloom ◽  
Kelly K. Lee
Keyword(s):  
Cell Culture ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Influenza Virus Infection ◽  
Sequence Diversity ◽  
Virus Protein

ABSTRACTInfluenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCEThe M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein’s function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1’s tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

scholarly journals The Hypervariable Immunodominant NP418-426 Epitope from the Influenza A Virus Nucleoprotein Is Recognized by Cytotoxic T Lymphocytes with High Functional Avidity

2006 ◽  
Vol 80 (12) ◽  
pp. 6024-6032 ◽  
Author(s):  
Adrianus C. M. Boon ◽  
Gerrie de Mutsert ◽  
Ron A. M. Fouchier ◽  
Albert D. M. E. Osterhaus ◽  
Guus F. Rimmelzwaan
Keyword(s):  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Influenza A ◽  
Mononuclear Cells ◽  
Influenza Viruses ◽  
Functional Avidity ◽  
Human Virus ◽  
Ctl Epitopes ◽  
Infected Cells

ABSTRACT Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A- and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP418-426 epitope was significantly higher than that of CTL recognizing other epitopes (P < 0.01). It was speculated that the high functional avidity of NP418-426-specific CTL was responsible for the diversification of this influenza A virus CTL epitope.

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