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Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN -β and IFN -λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding. IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies., (Copyright © 2018 American Society for Microbiology.)

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) causes a recently emerged human disease associated with pneumonia. The 5' end two-thirds of the single-stranded positive-sense viral genomic RNA, gene 1, encodes 16 mature proteins. Expression of nsp1, the most N-terminal gene 1 protein, prevented Sendai virus-induced endogenous IFN-beta mRNA accumulation without inhibiting dimerization of IFN regulatory factor 3, a protein that is essential for activation of the IFN-beta promoter. Furthermore, nsp1 expression promoted degradation of expressed RNA transcripts and host endogenous mRNAs, leading to a strong host protein synthesis inhibition. SCoV replication also promoted degradation of expressed RNA transcripts and host mRNAs, suggesting that nsp1 exerted its mRNA destabilization function in infected cells. In contrast to nsp1-induced mRNA destablization, no degradation of the 28S and 18S rRNAs occurred in either nsp1-expressing cells or SCoV-infected cells. These data suggested that, in infected cells, nsp1 promotes host mRNA degradation and thereby suppresses host gene expression, including proteins involved in host innate immune functions. SCoV nsp1-mediated promotion of host mRNA degradation may play an important role in SCoV pathogenesis.

Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles. An interaction between Gn and viral ribonucleoprotein complexes, including the direct binding of Gn to viral RNAs, drives viral RNA packaging into RVFV particles. To understand the mechanism of efficient antigenomic S RNA packaging in RVFV, we identified the regions in viral RNAs that directly interact with Gn by performing UV-crosslinking and immunoprecipitation of RVFV- infected cell lysates with anti-Gn antibody followed by high-throughput sequencing analysis (CLIP-seq analysis). Our data suggested the presence of multiple Gn-binding sites in RVFV RNAs, including a prominent Gn-binding site within the 3’ noncoding region of the antigenomic S RNA. We found that the efficient packaging of antigenomic S RNA was abrogated in a RVFV mutant lacking a part of this prominent Gn-binding site within the 3’ noncoding region. Also, the mutant RVFV, but not the parental RVFV, triggered the early induction of interferon-b mRNA expression after infection. These data suggest that the direct binding of Gn to the RNA element within the 3’ noncoding region of the antigenomic S RNA promoted the efficient packaging of antigenomic S RNA into virions. Furthermore, the efficient packaging of antigenomic S RNA into RVFV particles, driven by the RNA element, facilitated the synthesis of viral mRNA encoding NSs immediately after infection, resulting in the suppression of interferon-b mRNA expression. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the African continent and Middle East. RVFV NSs protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. These include suppression of general transcription, inhibition of IFN-β promoter induction and degradation of double-stranded RNA-dependent protein kinase R. Although each of these biological functions of NSs are considered important for countering the antiviral response in the host, the individual contributions of these functions towards RVFV virulence remains unclear. To examine this, we generated two RVFV MP-12 strain-derived mutant viruses. Each carried mutations in NSs that specifically targeted its general transcription inhibition function without affecting its ability to degrade PKR and inhibit IFN-β promoter induction, through its interaction with Sin3-associated protein 30, a part of the repressor complex at the IFN-β promoter. Using these mutant viruses, we have dissected the transcription inhibition function of NSs and examined its importance in RVFV virulence. Both NSs mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with MP-12. It has been reported that NSs suppresses general transcription by interfering with the formation of the transcription factor IIH complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. Our study results lead us to suggest that the ability of NSs to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. Importantly, RVFV MP-12-NSs mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus MP-12, highlighting the contribution of NSs-mediated general transcription inhibition towards RVFV virulence. [ABSTRACT FROM AUTHOR]

Abstract: Mouse embryonic fibroblasts (MEFs) are the most commonly used feeder cells for pluripotent stem cells. However, autogeneic feeder (AF) cells have several advantages such as no xenogeneic risks and reduced costs. In this report, we demonstrate that common marmoset embryonic stem (cmES) cells can be maintained on common marmoset AF (cmAF) cells. These cmES cells were maintained on cmAF cells for 6months, retaining their morphology, normal karyotype, and expression patterns for the pluripotent markers Oct-3/4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, as well as their ability to differentiate into cardiac and neural cells. Antibody array analysis revealed equivalent protein expression profiles between cmES cells maintained on cmAF cells and MEFs. In addition, similarly prepared human embryonic stem (hES) and induced pluripotent stem (hiPS) cell-derived AF cells supported the growth of and maintained the morphology and pluripotent marker expressions of hES and hiPS cells, respectively. DNA microarray analysis revealed that these hES and hiPS cells had mRNA expression profiles similar to those of hES and hiPS cells maintained on MEFs, respectively. Taken together, these findings imply that AF cells can replace MEFs in the routine maintenance of primate pluripotent stem cells. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the molecular and functional role of Zac1, a zinc finger-type transcription factor, as a cardiac transcription factor. The study conducted an analysis of the Zac1 mutant mice which reveals that Zac1 is needed for the cardiac morphological development and expression of genes, and Zac1 also acts as a transcriptional activator of cardiac genes. The study concludes that Zac1 has an essential role in the regulation of cardiac genes.

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression, including type I interferon production, by promoting host mRNA degradation and inhibiting host translation, in infected cells. We present evidence that nsp1 uses a novel, two-pronged strategy to inhibit host translation and gene expression. Nsp1 bound to the 40S ribosomal subunit and inactivated the translational activity of the 40S subunits. Furthermore, the nsp1–40S ribosome complex induced the modification of the 5′ region of capped mRNA template and rendered the template RNA translationally incompetent. Nsp1 also induced RNA cleavage in templates carrying the internal ribosome entry site (IRES) from encephalomyocarditis virus, but not in those carrying IRES elements from hepatitis C or cricket paralysis viruses, demonstrating that the nsp1-induced RNA modification was template-dependent. We speculate that the mRNAs that underwent the nsp1-mediated modification are marked for rapid turnover by the host RNA degradation machinery. [ABSTRACT FROM AUTHOR]

Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b) are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required. [ABSTRACT FROM AUTHOR]

Many viruses disrupt host gene expression by degrading host mRNAs and/or manipulating translation activities to create a cellular environment favorable for viral replication. Often, virus-induced suppression of host gene expression, including those involved in antiviral responses, contributes to viral pathogenicity. Accordingly, clarifying the mechanisms of virus-induced disruption of host gene expression is important for understanding virus–host cell interactions and virus pathogenesis. Three highly pathogenic human coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, have emerged in the past two decades. All of them encode nonstructural protein 1 (nsp1) in their genomes. Nsp1 of SARS-CoV and MERS-CoV exhibit common biological functions for inducing endonucleolytic cleavage of host mRNAs and inhibition of host translation, while viral mRNAs evade the nsp1-induced mRNA cleavage. SARS-CoV nsp1 is a major pathogenic determinant for this virus, supporting the notion that a viral protein that suppresses host gene expression can be a virulence factor, and further suggesting the possibility that SARS-CoV-2 nsp1, which has high amino acid identity with SARS-CoV nsp1, may serve as a major virulence factor. This review summarizes the gene expression suppression functions of nsp1 of CoVs, with a primary focus on SARS-CoV nsp1 and MERS-CoV nsp1. [ABSTRACT FROM AUTHOR]

The recent emergence of two highly pathogenic human coronaviruses (CoVs), severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, has ignited a strong interest in the identification of viral factors that determine the virulence and pathogenesis of CoVs. The nonstructural protein 1 (nsp1) of CoVs has attracted considerable attention in this regard as a potential virulence factor and a target for CoV vaccine development because of accumulating evidence that point to its role in the downregulation of host innate immune responses to CoV infection. Studies have revealed both functional conservation and mechanistic divergence among the nsp1 of different mammalian CoVs in perturbing host gene expression and antiviral responses. This review summarizes the current knowledge about the biological functions of CoV nsp1 that provides an insight into the novel strategies utilized by this viral protein to modulate host and viral gene expression during CoV infection. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) causes mosquito-borne epidemic diseases in humans and livestock. The virus carries three RNA segments, L, M, and S, of negative or ambisense polarity. L protein, an RNA-dependent RNA polymerase, encoded in the L segment, and N protein, encoded in the S segment, exert viral RNA replication and transcription. Coexpression of N, hemagglutinin (HA)-tagged L, and viral minigenome resulted in minigenome replication and transcription, a finding that demonstrated HA-tagged L was biologically active. Likewise L tagged with green fluorescent protein (GFP) was biologically competent. Coimmunoprecipitation analysis using extracts from cells coexpressing HA-tagged L and GFP-tagged L showed the formation of an L oligomer. Bimolecular fluorescence complementation analysis and coimmunoprecipitation studies demonstrated the formation of an intermolecular L-L interaction through its N-terminal and C-terminal regions and also suggested an intramolecular association between the N-terminal and C-terminal regions of L protein. A biologically inactive L mutant, in which the conserved signature SDD motif was replaced by the amino acid residues GNN, exhibited a dominant negative phenotype when coexpressed with wild-type L in the minigenome assay system. Expression of this mutant L also inhibited viral gene expression in virus-infected cells. These data provided compelling evidence for the importance of oligomerization of RVFV L protein for its polymerase activity. [ABSTRACT FROM AUTHOR]

We previously reported that transgenic (Tg) mice expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), were highly susceptible to SARS-CoV infection, which resulted in the development of disease of various severity and even death in some lineages. In this study, we further characterized and compared the pathogeneses of SARS-CoV infection in two of the most stable Tg lineages, AC70 and AC22, representing those susceptible and resistant to the lethal SARS-CoV infection, respectively. The kinetics of virus replication and the inflammatory responses within the lungs and brains, as well as the clinical and pathological outcomes, were assessed in each lineage. In addition, we generated information on lymphocyte subsets and mitogen-mediated proliferation of splenocytes. We found that while both lineages were permissive to SARS-CoV infection, causing elevated secretion of many inflammatory mediators within the lungs and brains, viral infection appeared to be more intense in AC70 than in AC22 mice, especially in the brain. Moreover, such infection was accompanied by a more profound immune suppression in the former, as evidenced by the extensive loss of T cells, compromised responses to concanavalin A stimulation, and absence of inflammatory infiltrates within the brain. We also found that CD8+ T cells were partially effective in attenuating the pathogenesis of SARS-CoV infection in lethality-resistant AC22 mice. Collectively, our data revealed a more intense viral infection and immunosuppression in AC70 mice than in AC22 mice, thereby providing us with an immunopathogenic basis for the fatal outcome of SARS-CoV infection in the AC70 mice. [ABSTRACT FROM AUTHOR]

nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs. [ABSTRACT FROM AUTHOR]