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The SARS coronavirus protein nsp1 can suppress host gene expression at a post-transcriptional level, with previous work showing a reduction in mRNA abundance. Now a direct effect on protein synthesis is revealed, as nsp1 modifies transcripts and also inactivates the 40S ribosomal subunit. 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.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disease that can result in disability. Until now, there is no antiviral treatment against CHIKV, demonstrating that there is a need for development of new drugs. Studies have shown that thiosemicarbazones and their metal complexes possess biological activities, and their synthesis is simple, clean, versatile, and results in high yields. Here, we evaluated the mechanism of action (MOA) of a cobalt(III) thiosemicarbazone complex named [Co III (L 1 ) 2 ]Cl based on its in vitro potent antiviral activity against CHIKV previously evaluated (80% of inhibition on replication) . Furthermore, the complex has no toxicity in healthy cells, as confirmed by infecting BHK-21 cells with CHIKV- nanoluciferase in the presence of the compound, showing that [Co III (L 1 ) 2 ]Cl inhibited CHIKV infection with the selective index of 3.26. [Co III (L 1 ) 2 ]Cl presented a post-entry effect on viral replication, emphasized by the strong interaction of [Co III (L 1 ) 2 ]Cl with CHIKV non-structural protein 4 (nsP4) in the microscale thermophoresis assay, suggesting a potential mode of action of this compound against CHIKV. Moreover, in silico analyses by molecular docking demonstrated potential interaction of [Co III (L 1 ) 2 ]Cl with nsP4 through hydrogen bonds, hydrophobic and electrostatic interactions. The evaluation of ADME-Tox properties showed that [Co III (L 1 ) 2 ]Cl presents appropriate lipophilicity, good human intestinal absorption, and has no toxicological effect as irritant, mutagenic, reproductive, and tumorigenic side effects. Graphical abstract

Background One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to ( Flaviviridae ). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function. Methods We cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes. Results The DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes. Conclusions Our data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.

Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4 114–135 peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca 2+ -activated Cl − channel TMEM16A, and by inhibiting Na + absorption by the epithelial Na + channel ENaC and the Na + /glucose cotransporter SGLT1. Activation of secretion and inhibition of Na + absorption by NSP4 114–135 , respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4 114–135 for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy.

The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV) non-structural proteins (NSPs), which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection.

There is a growing body of studies that show the important role of NS3 protein from hepatitis C virus in fibrosis. However, mechanisms of the effects of this protein on immune modulation of stellate cells remain to be investigated. Herein, the effect of NS3 protein on the expression level of suppressor of cytokine signaling (SOCS)1/3 and interleukin-24 (IL-24)-related genes was investigated in hepatic stellate cell (HSC), LX-2. Recombinant NS3 protein was added to LX-2 HSC culture. Leptin and standard medium treatments were also included in experiments as positive and negative controls, respectively. Total RNA was extracted from each well at 6, 12, and 24 h after NS3 addition. The expression levels of the fibrotic (transforming growth factor beta 1 [TGF-β], alpha-smooth muscle actin [α-SMA], and COL1A1), inflammatory (IL-6 and IL-24), IL-20R, IL-22R, and immunosuppressive genes (SOCS1 and SOCS3) were evaluated by real-time polymerase chain reaction (PCR). Recombinant NS3 protein induced activated phenotypes of LX-2 with a significant increase in the expression level of α-SMA COL1A1 (p < 0.0001) and TGF-β. Moreover, this exposure led to a meaningful elevation in the expression of IL-6. Furthermore, compared with leptin (control), after the stellate cell treatment with NS3, SOCS1 and SOCS3 gene expression induced at a comparable level. Compared with the control sample, the NS3 protein significantly increased the expression level of IL-24 and its related receptors, IL-20R and IL-22R. This study not only confirmed the previously proved inflammatory and fibrotic effect of this protein but also indicated that high expression levels of SOCS1, SOCS3, and IL-24 have a significant effect on HSC activation. Therefore, these two molecules can be used as a potential therapeutic target candidate.

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic α-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4⁺ T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG- and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAF(II)32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAF(II)32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.

Background Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Results Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity ( F  = 40.87, P  < 0.01); increased nuclear translocation of β-catenin ( F  = 165.26, P  < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression ( P  < 0.01); and promoted proliferation of Huh7 cells ( P  < 0.01 or P  < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells ( F  = 0.65, P  > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity ( F  = 64.25, P  < 0.01), and promoted the nuclear translocation of β-catenin ( F  = 66.54, P  < 0.01) and the Wnt3a-induced proliferation of LO2 cells ( P  < 0.01 or P  < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B ( P  < 0.01 or P  < 0.05). Conclusions HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

Persistent infection with GBV-C (GB Virus C), a non-pathogenic virus related to hepatitis C virus (HCV), prolongs survival in HIV infection. Two GBV-C proteins, NS5A and E2, have been shown previously to inhibit HIV replication in vitro. We investigated whether the GBV-C NS3 serine protease affects HIV replication. GBV-C NS3 protease expressed in a human CD4+ T lymphocyte cell line significantly inhibited HIV replication. Addition of NS4A or NS4A/4B coding sequence to GBV-C NS3 increased the effect on HIV replication. Inhibition of HIV replication was dose-dependent and was not mediated by increased cell toxicity. Mutation of the NS3 catalytic serine to alanine resulted in loss of both HIV inhibition and protease activity. GBV-C NS3 expression did not measurably decrease CD4 or CXCR4 expression. GBV-C NS3 serine protease significantly inhibited HIV replication without decreasing HIV receptor expression. The requirement for an intact catalytic serine at the active site indicates that inhibition was mediated by proteolytic cleavage of an unidentified target(s).