scholarly journals Association of Influenza Virus Matrix Protein with Ribonucleoproteins

1999 ◽  
Vol 73 (9) ◽  
pp. 7467-7473 ◽  
Author(s):  
Zhiping Ye ◽  
Teresa Liu ◽  
Daniel P. Offringa ◽  
Jonathan McInnis ◽  
Roland A. Levandowski
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Influenza A ◽  
Matrix Protein ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Zinc Finger Motif ◽  
Basic Amino Acids ◽  
Binding Regions

ABSTRACT To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.

scholarly journals Effects of Basic Amino Acids and Their Derivatives on SARS-CoV-2 and Influenza-A Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1301
Author(s):  
Ivonne Melano ◽  
Li-Lan Kuo ◽  
Yan-Chung Lo ◽  
Po-Wei Sung ◽  
Ni Tien ◽  
...  
Keyword(s):  
Amino Acids ◽  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Enveloped Viruses ◽  
Virus Attachment ◽  
Basic Amino Acids ◽  
Ester Derivative ◽  
Endosomal Acidification

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.

scholarly journals Restriction of Viral Replication by Mutation of the Influenza Virus Matrix Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 13055-13061 ◽  
Author(s):  
Teresa Liu ◽  
Zhiping Ye
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Zinc Finger ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Viral Assembly ◽  
Zinc Finger Motif

ABSTRACT The matrix protein (M1) of influenza virus plays an essential role in viral assembly and has a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and nucleoprotein not only promotes formation of helical RNP but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 in viral assembly and replication, mutations in the coding sequences of RKLKR and the zinc finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that the RKLKR domain of M1 protein plays an important role in viral replication.

scholarly journals Identification and Characterization of Heparin Binding Regions of the Fim2 Subunit of Bordetella pertussis

1998 ◽  
Vol 66 (5) ◽  
pp. 2256-2263 ◽  
Author(s):  
Cecile A. W. Geuijen ◽  
Rob J. L. Willems ◽  
Peter Hoogerhout ◽  
Wouter C. Puijk ◽  
Rob H. Meloen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bordetella Pertussis ◽  
Sequence Similarity ◽  
Basic Amino Acid ◽  
Cross Reactivity ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Basic Amino Acids ◽  
Binding Regions

ABSTRACT Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA

1991 ◽  
Vol 69 (9) ◽  
pp. 649-654 ◽  
Author(s):  
Christian Zwieb
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Site Directed Mutagenesis ◽  
Signal Recognition ◽  
C Terminus ◽  
Terminal Amino ◽  
Rna Complexes ◽  
Mutant Polypeptides

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein–RNA complexes on DEAE–Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136–144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113–120; they are suggested to be involved in interaction with signal recognition particle RNA.Key words: signal recognition particle, site-directed mutagenesis, protein–RNA binding.

scholarly journals Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

scholarly journals Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

2020 ◽  
pp. jbc.RA120.015524
Author(s):  
Alexandra P Breslawec ◽  
Shaochi Wang ◽  
Crystal Li ◽  
Myles B Poulin
Keyword(s):  
Amino Acids ◽  
Bacterial Infections ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Binding Surface ◽  
Activity Measurements ◽  
Rate Of Hydrolysis ◽  
Biofilm Dispersal ◽  
Hydrolysis Of

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the –1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

scholarly journals In vivo anti-influenza virus activity of a zinc finger peptide.

1997 ◽  
Vol 41 (3) ◽  
pp. 687-692 ◽  
Author(s):  
A K Judd ◽  
A Sanchez ◽  
D J Bucher ◽  
J H Huffman ◽  
K Bailey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Virus Infection ◽  
Zinc Finger ◽  
Matrix Protein ◽  
H3n2 Virus ◽  
Arterial Oxygen ◽  
Treatment Schedule ◽  
Zinc Finger Peptide

Matrix protein (M1) is a major structural protein of influenza virus, and it inhibits its own polymerase. A 19-amino-acid peptide, corresponding to a zinc finger region of the M1 sequence of influenza virus strain A/PR/8/34 (H1N1), centered around amino acids 148 to 166, was synthesized. This peptide, designated peptide 6, represents a zinc finger which includes a 7-amino-acid loop or finger and a 4-amino-acid tail at the carboxyl terminus, in addition to the 8 amino acids involved in the coordination of Zn. Three experiments were run to evaluate the activity of peptide 6 on infections induced in mice by influenza A/PR/8/34 and A/Victoria/3/75 (H3N2) viruses. Intranasal (i.n.) treatment of the H1N1 virus infection with 30 or 60 mg/kg of body weight/day, three times daily for 5 days, beginning 4 h pre-or 8 h post-virus exposure, was effective in preventing death, reducing the arterial oxygen decline, and inhibiting lung consolidation. Virus titers in the lungs determined on day 5 were reduced by up to 1.5 log10 in treated groups, but considerable variation in the titers of the recovered virus was seen. The H3N2 virus infection was treated i.n. with 30, 60, or 120 mg of peptide 6/kg/day by using the above-mentioned delayed initiation treatment schedule, and similar protection was seen, although lung virus titers were not reduced in the day-5 assay. Peptide 6 was well tolerated at doses up to 60 mg/kg/day. This zinc finger peptide may provide a new class of antivirals effective against influenza virus.

scholarly journals Antiviral Activity of Benzoic Acid Derivative NC-5 Against Influenza A Virus and Its Neuraminidase Inhibition

2019 ◽  
Vol 20 (24) ◽  
pp. 6261
Author(s):  
Min Guo ◽  
Jiawei Ni ◽  
Jie Yu ◽  
Jing Jin ◽  
Lingman Ma ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Benzoic Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Influenza Viruses ◽  
Drug Candidate ◽  
Dependent Manner ◽  
Drug Resistant

The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 μM and 32.8 μM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 μM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.

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