Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein.
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ABSTRACT:
SUBMITTER: Li Z
PROVIDER: S-EPMC2668439 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.
| S-EPMC103939 | biostudies-literature
Project description:Rift Valley fever phlebovirus (RVFV; Phenuiviridae, Phlebovirus) is an important mosquito-borne pathogen of both humans and ruminants. The RVFV genome is composed of tripartite, single stranded, negative or ambisense RNAs. The small (S) segment encodes both the nucleocapsid protein (N) and the non-structural protein (NSs). The N protein is responsible for the formation of the viral ribonucleoprotein (RNP) complexes, which are essential in the virus life cycle and for the transcription and replication of the viral genome. There is currently limited knowledge surrounding the roles of the RVFV nucleocapsid protein in viral infection other than its key functions: N protein multimerisation, encapsidation of the RNA genome and interactions with the RNA-dependent RNA polymerase, L. By bioinformatic comparison of the N sequences of fourteen phleboviruses, mutational analysis, minigenome assays and packaging assays, we have further characterised the RVFV N protein. Amino acids P11 and F149 in RVFV N play an essential role in the function of RNPs and are neither associated with N protein multimerisation nor known nucleocapsid protein functions and may have additional roles in the virus life cycle. Amino acid Y30 exhibited increased minigenome activity despite reduced RNA binding capacity. Additionally, we have determined that the N-terminal arm of N protein is not involved in N-L interactions. Elucidating the fundamental processes that involve the nucleocapsid protein will add to our understanding of this important viral protein and may influence future studies in the development of novel antiviral strategies.
| S-EPMC5764413 | biostudies-literature
Project description:Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions.
| S-EPMC5035998 | biostudies-literature
Project description:The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infection with the bat IAV H18N11. Yet, it has been difficult to biochemically determine the H18:MHC-II binding. Here, we followed a different approach and generated MHC-II chimeras from the human leukocyte antigen DR (HLA-DR), which supports H18-mediated entry, and the nonclassical MHC-II molecule HLA-DM, which does not. In this context, viral entry was supported only by a chimera containing the HLA-DR α1, α2, and β1 domains. Subsequent modeling of the H18:HLA-DR interaction identified the α2 domain as central for this interaction. Further mutational analyses revealed highly conserved amino acids within loop 4 (N149) and β-sheet 6 (V190) of the α2 domain as critical for virus entry. This suggests that conserved residues in the α1, α2, and β1 domains of MHC-II mediate H18-binding and virus propagation. The conservation of MHC-II amino acids, which are critical for H18N11 binding, may explain the broad species specificity of this virus.
| S-EPMC10325068 | biostudies-literature
Project description:BACKGROUND:Nucleoprotein (NP) of influenza viruses is utilized to differentiate between the A, B, and C viral serotypes. The availability of influenza genome sequence data has allowed us to identify specific amino acids at particular positions in viral proteins, including NP, known as "signature residues," which can be used to discriminate human influenza A viruses from H5N1 highly pathogenic avian influenza in human cases (HPAI) and pandemic H1N1(2009) (H1N1/2009) viruses. METHODS:Screening and epitope mapping of monoclonal antibodies (mAb) against NP of influenza A, which reacted differently with NP from human influenza A virus from HPAI and H1N1/2009 A virus. To identify the epitope(s) responsible for the discrimination of viral NP by mAbs, we prepared mutant NP proteins in the 293 cell expression system because some of the mAbs reacted with non-linear epitopes. RESULTS AND CONCLUSIONS:In the present study, we identified 3 mAbs. The results of epitope mapping showed that the epitopes were located at the signature residues. These results indicated that signature residues of NP could discriminate influenza A viruses from different origin.
| S-EPMC6505565 | biostudies-literature
Project description:Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.
| S-EPMC3610751 | biostudies-literature
Project description:The arenavirus nucleoprotein (NP) can suppress induction of type I interferon (IFN). This anti-IFN activity is thought to be shared by all arenaviruses with the exception of Tacaribe virus (TCRV). To identify the TCRV NP amino acid residues that prevent its IFN-countering ability, we created a series of NP chimeras between residues of TCRV NP and Pichinde virus (PICV) NP, an arenavirus NP with potent anti-IFN function. Chimera NP analysis revealed that a minimal four amino acid stretch derived from PICV NP could impart efficient anti-IFN activity to TCRV NP. Strikingly, the TCRV NP gene cloned and sequenced from viral stocks obtained through National Institutes of Health Biodefense and Emerging Infections (BEI) resources deviated from the reference sequence at this particular four-amino acid region, GPPT (GenBank KC329849) versus DLQL (GenBank NC004293), respectively at residues 389-392. When efficiently expressed in cells through codon-optimization, TCRV NP containing the GPPT residues rescued the antagonistic IFN function. Consistent with cell expression results, TCRV infection did not stimulate an IFNβ response early in infection in multiple cells types (e.g. A549, P388D1), and IRF-3 was not translocated to the nucleus in TCRV-infected A549 cells. Collectively, these data suggest that certain TCRV strain variants contain the important NP amino acids necessary for anti-IFN activity.
| S-EPMC3605688 | biostudies-literature
Project description:Influenza 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.IMPORTANCE The 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.
| S-EPMC6580950 | biostudies-literature
Project description:Influenza virus strains are often pleiomorphic, a characteristic that is largely attributed to specific residues in matrix protein 1 (M1). Although the mechanism by which M1 controls virion morphology has not yet been defined, it is suggested that the M1 interaction with other viral proteins plays an important role. In this study, we rescued recombinant virus WSN-AichiM1 containing the spherical A/WSN/33 (WSN) backbone and the M1 protein from A/Aichi/2/68 (Aichi). Aichi M1 differs from WSN M1 by 7 amino acids but includes those identified to be responsible for filamentous virion formation. Interestingly, Aichi virus produced spherical virions, while WSN-AichiM1 exhibited a long filamentous morphology, as detected by immunofluorescence and electron microscopy. Additional incorporation of Aichi nucleoprotein (NP) but not the hemagglutinin (HA), neuraminidase (NA), or M2 gene to WSN-AichiM1 abrogated filamentous virion formation, suggesting that specific M1-NP interactions affect virion morphology. Further characterization of viruses containing WSN/Aichi chimeric NPs identified residues 214, 217, and 253 of Aichi NP as necessary and sufficient for the formation of spherical virions. NP residues 214 and 217 localize at the minor groove between the two opposite-polarity NP helical strands of viral ribonucleocapsids, and residue 253 also localizes near the surface of the groove. These findings indicate that NP plays a critical role in influenza virus morphology, possibly through its interaction with the M1 layer during virus budding.
| S-EPMC3911569 | biostudies-literature
Project description:Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Å resolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73-90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.
| S-EPMC8248822 | biostudies-literature