NSP Gene Research Articles

Role of microRNA miR171 in plant development.

MicroRNAs (miRNAs) are endogenous non-coding small RNA with 19-24 nucleotides (nts) in length, which play an essential role in regulating gene expression at the post-transcriptional level. As one of the first miRNAs found in plants, miR171 is a typical class of conserved miRNAs. The miR171 sequences among different species are highly similar, and the vast majority of them have both "GAGCCG" and "CAAUAU" fragments. In addition to being involved in plant growth and development, hormone signaling and stress response, miR171 also plays multiple and important roles in plants through interactions with microbe and other small-RNAs. The miRNA functions by regulating the expression of target genes. Most of miR171's target genes are in the GRAS gene family, but also include some NSP, miRNAs, lncRNAs, and other genes. This review is intended to summarize recent updates on miR171 regarding its function in plant life and hopefully provide new ideas for understanding miR171 function and regulatory mechanisms.

Alphavirus Evasion of Zinc Finger Antiviral Protein (ZAP) Correlates with CpG Suppression in a Specific Viral nsP2 Gene Sequence.

Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effectors, including zinc finger antiviral protein (ZAP). Here, we demonstrated that Old World alphaviruses show differential sensitivity to endogenous ZAP in 293T cells: Ross River virus (RRV) and Sindbis virus (SINV) are more sensitive to ZAP than o'nyong'nyong virus (ONNV) and CHIKV. We hypothesized that the more ZAP-resistant alphaviruses evade ZAP binding to their RNA. However, we did not find a correlation between ZAP sensitivity and binding to alphavirus genomic RNA. Using a chimeric virus, we found the ZAP sensitivity determinant lies mainly within the alphavirus non-structural protein (nsP) gene region. Surprisingly, we also did not find a correlation between alphavirus ZAP sensitivity and binding to nsP RNA, suggesting ZAP targeting of specific regions in the nsP RNA. Since ZAP can preferentially bind CpG dinucleotides in viral RNA, we identified three 500-bp sequences in the nsP region where CpG content correlates with ZAP sensitivity. Interestingly, ZAP binding to one of these sequences in the nsP2 gene correlated to sensitivity, and we confirmed that this binding is CpG-dependent. Our results demonstrate a potential strategy of alphavirus virulence by localized CpG suppression to evade ZAP recognition.

Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells.

ABSTRACTSevere acute respiratory syndrome coronavirus (SARS-CoV)-2 is responsible for the COVID-19 pandemic that has caused unprecedented loss of life and economic trouble all over the world, though the mechanism of its replication remains poorly understood. In this study, antibodies were generated and used to systematically determine the expression profile and subcellular distribution of 11 SARS-CoV-2 nonstructural replicase proteins (nsp1, nsp2, nsp3, nsp5, nsp7, nsp8, nsp9, nsp10, nsp13, nsp14, and nsp15) by Western blot and immunofluorescence assay. Nsp3, nsp5, and nsp8 were detected in perinuclear foci at different time points, with diffusion and stronger fluorescence observed over time. In particular, colocalization of nsp8 and nsp13 with different replicase proteins suggested viral protein-protein interaction, which may be key to understanding their functions and potential molecular mechanisms. Viral intermediate dsRNA was detected in perinuclear foci as early as 2-h postinfection, indicating the initiation of virus replication. With the passage of time, these perinuclear dsRNA foci became larger and brighter, and nearly all colocalized with N protein, consistent with viral growth over time. Thus, the development of these anti-nsp antibodies provides basic tools for the further study of replication and diagnosis of SARS-CoV-2.IMPORTANCE The intracellular localization of SARS-CoV-2 replicase nonstructural proteins (nsp) during infection has not been fully elucidated. In this study, we systematically analyzed the expression and subcellular localization of 11 distinct viral nsp and dsRNA over time in SARS-CoV-2-infected cells by using individual antibody against these replicase proteins. The data indicated that nsp gene expression is highly regulated in space and time, which could be useful to understand the function of viral replicases and future development of diagnostics and potential antiviral strategies against SARS-CoV-2.

NSP protein encoded in negative NS RNA strand of influenza A virus induces cellular immune response in infected animals

Infection of mice with influenza A viruses led to the formation of clones of lymphocytes that specifically recognizes viral domains in the central zone of the NSP protein (amino acid positions 83-119). Computer analysis of the primary structure of the NSP protein showed the presence of T-cell epitopes in the central part of the NSP molecule. The findings indicate that the viral NSP gene is expressed in the infected animals and support the concept of the bipolar strategy (ambisense strategy) of the influenza A virus genome.

NSP Protein Encoded in Negative NS RNA Strand of Influenza A Virus Induces Cellular Immune Response in Infected Animals.

Infection of mice with influenza A viruses led to the formation of clones of lymphocytes that specifically recognizes viral domains in the central zone of the NSP protein (amino acid positions 83-119). Computer analysis of the primary structure of the NSP protein showed the presence of T-cell epitopes in the central part of the NSP molecule. The findings indicate that the viral NSP gene is expressed in the infected animals and verify the concept of the bipolar strategy (ambisense strategy) of the influenza A virus genome.

Recombinant Tomato leaf curl New Delhi virus is associated with yellow vein mosaic disease of okra in India

Okra is the important vegetable crop grown across tropical and subtropical regions of the world. Yellow vein mosaic (YVMD) and enation leaf curl (ELCuD) diseases caused by begomoviruses are the major threat for cultivation and production of okra in India. Survey conducted between 2006 and 2010 revealed the incidence of begomovirus diseases ranged from 55 to 90% in okra growing states (ten states and two union territories) of the country. Three hundred forty-three okra leaf samples showing symptoms such as yellow mosaic, vein thickening, petiole bending, complete yellowing and upward leaf curling of leaves were collected from the surveyed locations across India. The PCR detection for begomovirus in the okra samples and subsequent cloning of partial genome sequencing of DNA A (1.2 kb fragment) indicated, 290 samples are associated with monopartite virus and 53 samples were associated with bipartite virus. Among the isolates showing highest identity with ToLCNDV, one isolate from each from five states was selected for amplification of full-length genomes (both DNA A and DNA B). The sequence analysis of DNA A and B components of five isolates revealed presence of nucleotide (nt) identity of >90% with ToLCNDV infecting tomato, eggplant, and potato. Phylogenetic and recombination analysis indicated that both DNA A and DNA B components of ToLCNDV infecting okra has the recombinant origin from BYVMV, ToLCNDV, and SLCCNV as the foremost parents. Further, dot blot hybridization for detection of ToLCNDV was developed for using CP and NSP gene probe. This is the first report of ToLCNDV associated with yellow vein mosaic disease of okra in India.

Phylogenetic analysis of human G9P[8] rotavirus strains circulating in Jiangsu, China between 2010 and 2016.

Rotavirus A (RVA) is the leading cause of acute viral gastroenteritis in children under 5 years of age worldwide. G9P[8] is a common RVA genotype that has been persistently prevalent in Jiangsu, China. To determine the genetic diversity of G9P[8] RVAs, 7 representative G9P[8] strains collected from Suzhou Children's Hospital between 2010 and 2016 (named JS2010-JS2016) were analyzed through whole-genome sequencing. All evaluated strains showed the Wa-like constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Furthermore, phylogenetic analysis revealed that the VP7 genes of all strains clustered into lineage G9-III and G9-VI. With the exception of strain JS2012 (P[8]-4), the VP4 sequences of all strains belonged to the P[8]-3 lineage. Sequencing further revealed that amino acid substitutions were present in the antigenic regions of the VP7 and VP4 genes of all strains. Moreover, there were multiple substitutions in antigenic sites I and II of the nonstructural protein 4 (NSP4) genes, whereas the other NSP genes were relatively conserved. In conclusion, our phylogenetic analysis of these 7 G9P[8] strains suggests that RVA varied across regions and time. Therefore, our findings suggest that continued surveillance is necessary to explore the molecular evolutionary characteristics of RVA for better prevention and treatment of acute viral gastroenteritis.

Detection of rabbit haemorrhagic disease virus 2 (GI.2) in Poland.

In this paper we present the first cases of rabbit haemorrhagic disease virus 2 (RHDV2 - GI.2) in Poland. The virus was detected in liver samples of RHD-suspected rabbits from Lodzkie and west Pomeranian voivodeships. In both cases, the typical clinical symptoms of the disease were observed despite the fact that the rabbits were previously vaccinated against RHD. In order to extend the analysis of the RHDV2 strain infecting the rabbits, the entire VP60 and NSP genes were amplified and sequenced. The results of rRT-PCR assay have shown that tested RHDV samples were positive for the presence of RHDV2. In the phylogenetic analysis of vp60gene the first Polish RHDV isolates (RED 2016 and VMS 2017) clustered together with the reference RHDV2, meaning they represent new evolutionary RHDV linkeages. The first Polish RHDV2 isolates showed about 97% nucleotide sequence identity with the reference RHDV2 strains and approximately 18% difference from classic RHDV and RHDVa variants.

Second‐generation sequencing for gene discovery in the Brassicaceae

The Brassicaceae contains the most diverse collection of agriculturally important crop species of all plant families. Yet, this is one of the few families that do not form functional symbiotic associations with mycorrhizal fungi in the soil for improved nutrient acquisition. The genes involved in this symbiosis were more recently recruited by legumes for symbiotic association with nitrogen-fixing rhizobia bacteria. This study applied second-generation sequencing (SGS) and analysis tools to discover that two such genes, NSP1 (Nodulation Signalling Pathway 1) and NSP2, remain conserved in diverse members of the Brassicaceae despite the absence of these symbioses. We demonstrate the utility of SGS data for the discovery of putative gene homologs and their analysis in complex polyploid crop genomes with little prior sequence information. Furthermore, we show how this data can be applied to enhance downstream reverse genetics analyses. We hypothesize that Brassica NSP genes may function in the root in other plant-microbe interaction pathways that were recruited for mycorrhizal and rhizobial symbioses during evolution.

Microevolutionary dynamics of a macroevolutionary key innovation in a Lepidopteran herbivore

BackgroundA molecular population genetics understanding is central to the study of ecological and evolutionary functional genomics. Population genetics identifies genetic variation and its distribution within and among populations, it reveals the demographic history of the populations studied, and can provide indirect insights into historical selection dynamics. Here we use this approach to examine the demographic and selective dynamics acting of a candidate gene involved in plant-insect interactions. Previous work documents the macroevolutionary and historical ecological importance of the nitrile-specifier protein (Nsp), which facilitated the host shift of Pieridae butterflies onto Brassicales host plants ~80 Myr ago.ResultsHere we assess the microevolutionary dynamics of the Nsp gene by studying the within and among-population variation at Nsp and reference genes in the butterfly Pieris rapae (Small Cabbage White). Nsp exhibits unexpectedly high amounts of amino acid polymorphism, unequally distributed across the gene. The vast majority of genetic variation exists within populations, with little to no genetic differentiation among four populations on two continents. A comparison of synonymous and nonsynonymous substitutions in 70 randomly chosen genes among P. rapae and its close relative Pieris brassicae (Large Cabbage White) finds Nsp to have a significantly relaxed functional constraint compared to housekeeping genes. We find strong evidence for a recent population expansion and no role for strong purifying or directional selection upon the Nsp gene.ConclusionsThe microevolutionary dynamics of the Nsp gene in P. rapae are dominated by recent population expansion and variation in functional constraint across the repeated domains of the Nsp gene. While the high amounts of amino acid diversity suggest there may be significant functional differences among allelic variants segregating within populations, indirect tests of selection could not conclusively identify a signature of historical selection. The importance of using this information for planning future studies of potential performance and fitness consequences of the observed variation is discussed.

Identification of an rsh gene from a Novosphingobium sp. necessary for quorum-sensing signal accumulation.

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

Molecular characterization of the 9.36 kb C-terminal region of canine coronavirus 1-71 strain

A total of 9.36 kb nucleotides of the C-terminal one-third genome of the canine Coronavirus (CCV) 1-71 strain, including all the structural protein genes and some non-structural protein (nsp) genes were cloned and sequenced. Nucleotide and amino acid sequence alignment as well as phylogenetic analysis of all the structural ORFs with reference coronavirus strains showed that CCV 1-71 was highly related to Chinese CCV isolates. This indicates that these CCV stains may have the same ancestor. Two large deletions were found in ORF3 and led to an elongated nsp3a and a truncated nsp3b. A single “A” insertion in a 6A stretch resulted in the truncation of nsp7b. The great variation in nsp3b and nsp7b indicates that these proteins are not essential for the viral replication.

Rat and chicken s-rex/ NSP mRNA: nucleotide sequence of main transcripts and expression of splice variants in rat tissues

Two main transcripts of the s-rex/ NSP gene are generated by different promoter usage and differential splicing in neuronal and endocrine tissues of higher vertebrates, suggesting that the encoded proteins function in neuroendocrine secretion. To know more about the structure, expression and evolution of this new gene, we have cloned full-length cDNAs for both 1.5 kb and 3.5 kb transcripts from rat and chicken brain cDNA libraries. Sequence analysis has revealed structures within the 3′- UTR that are conserved in these mRNAs and human NSP mRNA and that could be involved in specific compartmentalization of s-rex/ NSP mRNA in neuronal cells. An additional transcript generated by differential splicing of internal exons has been cloned from a rat DRG library. Low levels of s-rex/ NSP mRNAs have been detected in some non-neuroendocrine tissues, although substantial levels of a unique transcript have been found in rat testis. By RT-PCR analysis, other tissue-specific transcripts that are products of rare splicing events have been revealed.

NSP-encoded reticulons are neuroendocrine markers of a novel category in human lung cancer diagnosis: Van de Velde HJK, Senden NHM, Roskams TAD, Broers JLV, Ramaekers FCS, Roebroek A.J.M. et al. Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven, Herestraat 49, B-3000 Leuven. Cancer Res 1994;54:4769–4776

The NSP gene was recently shown to constitute the prototype of a novel gene family, to be selectively transcribed in neural and endocrine cells, and to encode three overlapping proteins, NSP-A, NSP-B, and NSP-C. These proteins were collectively designated reticulons, because they were found to be anchored to membranes of the endoplasmic reticulum through their common carboxy-terminal regions. The goal of the present study was to determine whether the reticulons might be used as markers for neuroendocrine differentiation in human lung tumors. Therefore, the tissue distribution of the NSP-A protein was studied and expression in human lung tumors was evaluated. Immunohistochemical analysis of normal tissues with monoclonal antibodies specifically recognizing the NSP-A protein indicated that NSP-A exhibits a distinct neuroendocrine distribution pattern since it was found to be expressed in a variety of cells with an established neuroendocrine phenotype but not in cells lacking such features. Results with specimens of a wide variety of primary human tumors provided further support for this claim. Immunohistochemical analysis of primary lung carcinomas revealed that NSP-A was readily detectable in small cell lung carcinoma (SCLCs) (8 of 12) and carcinoid tumors of the lung (3 of 3) but not in nonneuroendocrine non-SCLCs (0 of 10). In 13 of 27 non-SCLCs expressing the neural cell adhesion molecule and/or neurofilament proteins, however, NSP-A was found to be expressed. Northern blot analysis of human lung carcinoma cell lines revealed expression of NSP-A- and/or NSP-C-encoding mRNAs in all 18 SCLC cell lines that were studied, except one; however, no expression of these mRNAs could be detected in any of the 11 non-SCLC cell lines tested. The NSP transcript encoding NSP-B was found only in SCLC cell line NCI-H82. In conclusion, the results of our studies suggest that, in lung tumor cells, expression of NSP-A and most likely also NSP-C is restricted to cells with a neuroendocrine phenotype.

NSP gene expression as a novel marker of the neuroendocrine phenotype in lung carcinomas : H.J.K. van de Velde 1, N.H.M. Senden 2, A.J.M. Roebroek 1, J.L.V. Broers 2, F.C.S. Ramaekers 2 and W.J.M. Van de Ven 1 ( 1Lab. for Molecular Oncology, Center for Human Genetics, Univ. of Leuven, Belgium and 2Dept. of Molecular Cell Biology and Genetics, Univ.

NSP gene expression as a novel marker of the neuroendocrine phenotype in lung carcinomas : H.J.K. van de Velde 1, N.H.M. Senden 2, A.J.M. Roebroek 1, J.L.V. Broers 2, F.C.S. Ramaekers 2 and W.J.M. Van de Ven 1 ( 1Lab. for Molecular Oncology, Center for Human Genetics, Univ. of Leuven, Belgium and 2Dept. of Molecular Cell Biology and Genetics, Univ.

Molecular analysis of expression in rat brain of NSP-A, a novel neuroendocrine-specific protein of the endoplasmic reticulum

Previous studies have established that the novel neuroendocrine-specific NSP gene encodes three carboxy-terminally overlapping proteins, NSP-A, NSP-B and NSP-C, which are anchored to membranes of the endoplasmic reticulum. Here, we report results of studies in which expression of NSP-A in rat brain was investigated. Immunization of mice with a bacterial hybrid protein containing almost all NSP-A sequences led to the isolation of five monoclonal anti-NSP-A antibodies. The corresponding epitopes were found to be mapping to two regions unique to NSP-A. In Western blot analysis of rat cerebrum and cerebellum using these antibodies, proteins of about 145 kDa were detected. An immunohistochemical study of rat brain revealed the presence of NSP-A in many brain regions, particularly in cerebellar Purkinje cells, in neurons of the superior colliculus and of the pyriform and enthorhinal cortex, in fibers of the basal ganglia and several hippocampal regions including CA3 (stratum lucidum) and the dentate gyrus, in the induseum griseum and in the subcommissural organ, suggesting a role of NSP-A in many areas of the brain.

Regional mapping of the human NSP gene to chromosome region 14q21→q22 by fluorescence in situ hybridization analysis

Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were isolated from cDNA libraries constructed with mRNA isolated from human lung carcinoma cells. Hybridization analysis of a panel of human x mouse cell hybrids with an 0.8-kb NSP cDNA probe indicated that the human NSP gene is probably located on chromosome 14. Fluorescence in situ hybridization analysis of metaphase chromosomes using overlapping genomic clones of NSP as a probe localized the NSP gene to chromosome region 14q21-->q22.

Cloning and expression of alternative transcripts of a novel neuroendocrine-specific gene and identification of its 135-kDa translational product

Monoclonal antibodies RNL-2 and RNL-3 were previously shown to react with four 35-45-kDa proteins, expressed only in small cell lung carcinoma NCI-H82 cells, but to stain a subset of neuroendocrine tissues and neoplasms (Broers, J. L. V., Mijnheere, E. P., Klein Rot, M., Schaart, G., Sijlmans, A., Boerman, O. C., and Ramaekers, F. C. S. (1991) Cancer 67, 619-633). We used RNL-2 and RNL-3 to isolate cDNA sequences that code for proteins containing the two corresponding epitopes and utilized such cDNAs to develop second generation antibodies. Using these antibodies, we identified a novel 135-kDa protein. The corresponding cDNAs were found to belong to a previously unknown gene with a neuroendocrine-specific expression pattern, tentatively designated NSP gene. NSP transcription appeared to result in mRNAs of 3.4 and 1.8 kilobases (kb). In the NCI-H82 cells only, an apparently aberrant transcript of 2.3 kb was found. cDNAs containing coding sequences of the 3.4-, 2.3-, and 1.8-kb transcripts were isolated, and nucleotide sequence analysis revealed extensive sequence overlap and open reading frames for proteins of 776, 356, and 208 amino acids, respectively. The three deduced proteins all have a common carboxyl-terminal part with two large hydrophobic regions. Transfection of the complete coding sequences of the 3.4-kb transcript resulted in the production of a protein with an apparent molecular mass of 135 kDa. This protein is predicted to be highly negatively charged (calculated pI of 4.35), to be rich in proline and serine, and to contain multiple potential phosphorylation sites.