Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.
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ABSTRACT:
SUBMITTER: Waters K
PROVIDER: S-EPMC8579824 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter.MethodsUsing entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses.ResultsWe found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses.ConclusionsOur results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.
| S-EPMC6668090 | biostudies-literature
Project description:BackgroundThe first and only case of human infection with the avian influenza A (H7N4) virus in China emerged in 2018. The H7N4 virus was distinct from previous H7N9 viruses and raised public concerns. Therefore, developing a suitable H7N4 candidate vaccine virus (CVV) remains crucial for potential pandemic preparedness.MethodsWe constructed a reassortant virus with a (6 + 2) genome composition, then introduced the polymerase basic protein 1 (PB1) from a wild-type virus to develop a (5 + 3) reassortant virus through reverse genetics. We performed whole-genome sequencing to confirm the genome stability, assessed the growth ability in MDCK cells, and analyzed virus antigenicity using hemagglutination inhibition assays. Subsequently, the effect of homologous PB1 on polymerase activity, viral protein yield, and pathogenicity was assessed.ResultsThe (5 + 3) virus harbouring the homologous PB1 gene exhibited significantly improved growth characteristics, higher viral protein yield, and polymerase activity than the (6 + 2) virus. After successive passage in embryonated eggs, glutamic acid (E) substituted glycine(G) at position 218 (H3 numbering) in the hemagglutinin (HA) gene of both (5 + 3) and (6 + 2) viruses. The substitution improved the growth of the (6 + 2) virus but exhibited no significant effect or alteration on the antigenicity of the (5 + 3) virus. Moreover, the (5 + 3) virus exhibited low pathogenicity in chickens and ferrets.ConclusionHomologous PB1 of the H7N4 virus improves the growth ability while sustaining low pathogenicity. Collectively, the gene composition of the (5 + 3) reassortant virus is a suitable H7N4 CVV for potential pandemic preparedness.
| S-EPMC8983892 | biostudies-literature
Project description:DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.
| S-EPMC4000090 | biostudies-other
Project description:The glutaredoxin (Grx) family of oxidoreductases has a conserved residue at position 8 that varies between Arginine in Grx1 and Lysine in Grx3. It has been proposed that this Arg/Lys change is the main cause for the 35 mV difference in redox potential between the two enzymes. To gain insights into the catalytic machinery of Grx3 and directly evaluate the role of residue 8 in the catalysis of thiol-disulfide exchange by this enzyme, we synthesized the "wild type" enzyme (sGrx3), and four analogues substituting the lysine at position 8 with arginine, ornithine (Orn), citrulline (Cit) and norvaline (Nva). The redox potential and equilibration kinetics with thioredoxin (Trx1) were determined for each enzyme by fluorescence intensity. While minor effects on redox potential were observed, we found that residue 8 had a more marked effect on the catalytic efficiency of this enzyme. Surprisingly, truncation of the functional group resulted in a more efficient enzyme, Lys8Nva, exhibiting rate constants that are an order of magnitude higher than sGrx3 for both forward and reverse reactions. These observations pose the question why would a residue that reduces the rate of enzyme turnover be evolutionarily conserved? The significant changes in the kinetic parameters suggest that this position plays an important role in the thiol-disulfide exchange reaction by affecting the nucleophilic thiolate through electrostatic or hydrogen bonding interactions. Since the reduced Grx has an exposed thiol that could easily be alkylated, either Arg or Lys could act as a gatekeeper that deters unwanted electrophiles from attacking the active site thiolate.
| S-EPMC3820274 | biostudies-literature
Project description:SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.
| S-EPMC4558342 | biostudies-literature
Project description:The MYC gene encodes a human transcription factor and proto-oncogene that is dysregulated in over half of all known cancers. To better understand potential post-transcriptional regulatory features affecting MYC expression, we analyzed secondary structures in the MYC mRNA using a program that is optimized for finding small locally-folded motifs with a high propensity for function. This was accomplished by calculating folding metrics across the MYC sequence using a sliding analysis window and generating unique consensus base pairing models weighted by their lower-than-random predicted folding energy. A series of 30 motifs were identified, primarily in the 5' and 3' untranslated regions, which show evidence of structural conservation and compensating mutations across vertebrate MYC homologs. This analysis was able to recapitulate known elements found within an internal ribosomal entry site, as well as discover a novel element in the 3' UTR that is unusually stable and conserved. This novel motif was shown to affect MYC expression, potentially via the modulation of miRNA target accessibility or other trans-regulatory factors. In addition to providing basic insights into mechanisms that regulate MYC expression, this study provides numerous, potentially druggable RNA targets for the MYC gene, which is considered "undruggable" at the protein level.
| S-EPMC6576772 | biostudies-literature
Project description:Polysialic acid on the neural cell adhesion molecule (NCAM) modulates cell-cell adhesion and signaling, is required for proper brain development, and plays roles in neuronal regeneration and the growth and invasiveness of tumor cells. Evidence indicates that NCAM polysialylation is highly protein-specific, requiring an initial polysialyltransferase-NCAM protein-protein interaction. Previous work suggested that a polybasic region located prior to the conserved polysialyltransferase catalytic motifs may be involved in NCAM recognition, but not overall enzyme activity (Foley, D. A., Swartzentruber, K. G., and Colley, K. J. (2009) J. Biol. Chem. 284, 15505-15516). Here, we employ a competition assay to evaluate the role of this region in substrate recognition. We find that truncated, catalytically inactive ST8SiaIV/PST proteins that include the polybasic region, but not those that lack this region, compete with endogenous ST8SiaIV/PST and reduce NCAM polysialylation in SW2 small cell lung carcinoma cells. Replacing two polybasic region residues, Arg(82) and Arg(93), eliminates the ability of a full-length, catalytically inactive enzyme (PST H331K) to compete with SW2 cell ST8SiaIV/PST and block NCAM polysialylation. Replacing these residues singly or together in ST8SiaIV/PST substantially reduces or eliminates NCAM polysialylation, respectively. In contrast, replacing Arg(82), but not Arg(93), substantially reduces the ability of ST8SiaIV/PST to polysialylate neuropilin-2 and SynCAM 1, suggesting that Arg(82) plays a general role in substrate recognition, whereas Arg(93) specifically functions in NCAM recognition. Taken together, our results indicate that the ST8SiaIV/PST polybasic region plays a critical role in substrate recognition and suggest that different combinations of basic residues may mediate the recognition of distinct substrates.
| S-EPMC3307320 | biostudies-literature
Project description:BackgroundPlant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported.ResultsA phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein.ConclusionsThe bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence potentially constituting a major source of functional diversity among members of this subfamily.
| S-EPMC3060862 | biostudies-literature
Project description:UnlabelledThe replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.ImportanceLittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.
| S-EPMC4337532 | biostudies-literature
Project description:Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.
| S-EPMC6881253 | biostudies-literature