Insights into the role of the cobalt(III)-thiosemicarbazone complex as a potential inhibitor of the Chikungunya virus nsP4.

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 [CoIII(L1)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 [CoIII(L1)2]Cl inhibited CHIKV infection with the selective index of 3.26. [CoIII(L1)2]Cl presented a post-entry effect on viral replication, emphasized by the strong interaction of [CoIII(L1)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 [CoIII(L1)2]Cl with nsP4 through hydrogen bonds, hydrophobic and electrostatic interactions. The evaluation of ADME-Tox properties showed that [CoIII(L1)2]Cl presents appropriate lipophilicity, good human intestinal absorption, and has no toxicological effect as irritant, mutagenic, reproductive, and tumorigenic side effects.

Peptides derived from viral glycoprotein Gc Inhibit infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel phlebovirus (SFTSV), characterized by fever, thrombocytopenia and leukocytopenia which lead to multiple organ failure with high mortality in severe cases. The SFTSV has spread rapidly in recent years and posed a serious threat to public health in endemic areas. However, specific antiviral therapeutics for SFTSV infection are rare. In this study, we demonstrated that two peptides, SGc1 and SGc8, derived from a hydrophobic region of the SFTSV glycoprotein Gc, could potently inhibit SFTSV replication in a dose-dependent manner without apparent cytotoxicity in various cell lines and with low immunogenicity and good stability. The IC50 (50% inhibition concentration) values for both peptides to inhibit 2 MOI of SFTSV infection were below 10 µM in L02, Vero and BHK21 cells. Mechanistically, SGc1 and SGc8 mainly inhibited viral entry at the early stage of the viral infection. Inhibition of SFTSV replication was specific by both peptides because no inhibitory effect was shown against other viruses including Zika virus and Enterovirus A71. Taken together, our results suggested that viral glycoprotein-derived SGc1 and SGc8 peptides have antiviral potential and warrant further assessment as an SFTSV-specific therapeutic.

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Zika virus NS5 protein inhibits cell growth and invasion of glioma.

Glioma is the most common primary brain tumor with high mortality. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. Oncolytic viral therapy for glioma has developed as an exciting therapeutic method in recent years. Zika virus, a member of flavivirus family, has oncolytic activity against glioma cells but the mechanism is unknown. Here, we aimed to determine which viral protein might play a critical role in mitigating glioma cell growth. We examined the tumor suppressor function of four nonstructural proteins NS1, NS3, NS4B and NS5 in human glioma cell line U87. As a result, we found that only NS5 significantly inhibited proliferation, migration and invasion of U87 cells. Moreover, expression of NS5 suppressed tumorigenicity of mouse GL261 glioma cell in vivo. Our findings provide some clues for further exploration of oncolytic Zika virus in the treatment of glioma.

Recombinant NS3 Protein Induced Expression of Immune Modulatory Elements in Hepatic Stellate Cells During Its Fibrotic Activity.

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-ß], 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-ß. 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.

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/ß-catenin pathway.

BACKGROUND: Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/ß-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/ß-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 ß-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of ß-catenin (F = 165.26, P < 0.01); upregulated nuclear ß-catenin, cytoplasmic ß-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 ß-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced ß-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of ß-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/ß-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/ß-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.

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response.

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis.

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93-140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.

HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium.

Direct association of dry eye syndrome and hepatitis C virus (HCV) infection is a well established fact. In this context, the current study examines the in vitro corneal inflammatory response with respect to HCV core and NS3 antigens. Toll like receptors (TLRs) are pattern recognition receptors which can mediate innate immune response. In the present study, corneal epithelial cells responded to HCV core and NS3 proteins by secreting pro-inflammatory cytokines IL-8, IL-6 and TNF-α via TLR1, TLR2 and TLR6 mediated innate immune response. MyD88/NF-kB signalling was involved in pro-inflammatory cytokine production. Corneal epithelium synthesised nitric oxide (NO) via iNOS during HCV core and NS3 exposure. On later stages of inflammation, cells underwent apoptosis which lead to cell death. SiRNA mediated silencing of TLR1, TLR2 and TLR6 resulted in a significant down regulation of IL-8 and NO. In conclusion, this study indicates that HCV core and NS3 proteins are capable of inducing immune response in corneal epithelium which can potentiate the pathology of HCV associated dry eye condition. Blocking specific TLR response can have therapeutic application in controlling the inflammatory response associated with this dry eye condition.

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors.

UNLABELLED: Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. CONCLUSION: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment.

Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53.

p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

Screening and rational design of hepatitis C virus entry inhibitory peptides derived from GB virus A NS5A.

Chronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selected Flaviviridae viruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCV in vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

MEK5/ERK5/mef2: a novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.

Modulation of programmed cell death pathways by the hepatitis C virus.

Hepatitis C virus (HCV), a positive stranded RNA virus of the family Flaviviridae, is the major cause of non-A, non-B hepatitis worldwide. The HCV genome encodes a precursor polyprotein of ~ 3,000 amino acids that is processed co-translationally and post-translationally to give rise to viral structural and non-structural proteins. Nearly all of these viral proteins have been shown to modulate cell death via various mechanisms. In addition, studies using the replicon and recombinant HCV cell culture systems have yield important insights into the modulation of programmed cell death by HCV replication. Here, we summarize current knowledge on the modulation of apoptosis and other programmed cell death pathways by the HCV in these cell culture systems.

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-ß production by porcine reproductive and respiratory syndrome virus.

Induction of type I interferon (IFN-α/ß) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-ß production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-ß during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-ß and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.

The effects of hepatitis C virus non-structural protein 3 on cell growth mediated by extracellular signal-related kinase cascades in human hepatocytes in vitro.

Hepatitis C virus (HCV) infection has become a severe health problem worldwide. The viral proteins are believed to be among the most important factors that contribute to HCV mediated pathogenesis. Accumulated evidence demonstrating that HCV non-structural protein 3 (NS3) possesses oncogenic potential, and is involved in the regulation of cell proliferation has been documented. In this study, we emphasized the effect of HCV NS3 protein on cell proliferation in the immortally normal hepatocyte QSG7701 cells. The cell line transfected with plasmid expressing NS3 protein showed enhanced cell growth, extracellular signal-related kinase (ERK) activation, DNA binding activities of transcription factors of activator protein 1 (AP-1) and NF-kappaB, and cyclin D1 overexpression, but without activation of Jun amino-terminal kinase or p38. Pre-treatment of NS3 protein expressing cells with ERK inhibitor, PD98059, blocked the activation of AP-1 and NF-kappaB, and inhibited cyclin D1 expression and cell proliferation. The results suggest that NS3-mediated cell growth occurs through activation of ERK/AP-1 and NF-kappaB/cyclin D1 cascades.

Hepatitis C virus NS5A activates the mammalian target of rapamycin (mTOR) pathway, contributing to cell survival by disrupting the interaction between FK506-binding protein 38 (FKBP38) and mTOR.

Hepatitis C virus (HCV) often establishes a persistent infection that most likely involves a complex host-virus interplay. We previously reported that the HCV nonstructural protein 5A (NS5A) bound to cellular protein FKBP38 and resulted in apoptosis suppression in human hepatoma cell line Huh7. In the present research we further found that NS5A increased phosphorylation levels of two mTOR-targeted substrates, S6K1 and 4EBP1, in Huh7 in the absence of serum. mTOR inhibitor rapamycin or NS5A knockdown blocked S6K1 and 4EBP1 phosphorylation increase in NS5A-Huh7 and HCV replicon cells, suggesting that NS5A specifically regulated mTOR activation. Overexpression of NS5A and FKBP38 mutants or FKBP38 knockdown revealed this mTOR activation was dependent on NS5A-FKBP38 interaction. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 treatment in NS5A-Huh7 showed that the mTOR activation was independent of PI3K. Moreover, NS5A suppressed caspase 3 and poly(ADP-ribose) polymerase activation, which was abolished by NS5A knockdown or rapamycin, indicating NS5A inhibited apoptosis specifically through the mTOR pathway. Further analyses suggested that apoptotic inhibition exerted by NS5A via mTOR also required NS5A-FKBP38 interaction. Glutathione S-transferase pulldown and co-immunoprecipitation showed that NS5A disrupted the mTOR-FKBP38 association. Additionally, NS5A or FKBP38 mutants recovered the mTOR-FKBP38 interaction; this indicated that the impairment of mTOR-FKBP38 association was dependent on NS5A-FKBP38 binding. Collectively, our data demonstrate that HCV NS5A activates the mTOR pathway to inhibit apoptosis through impairing the interaction between mTOR and FKBP38, which may represent a pivotal mechanism for HCV persistence and pathogenesis.

Characterization of a peptide domain within the GB virus C NS5A phosphoprotein that inhibits HIV replication.

BACKGROUND: GBV-C infection is associated with prolonged survival in HIV-infected people and GBV-C inhibits HIV replication in co-infection models. Expression of the GBV-C nonstructural phosphoprotein 5A (NS5A) decreases surface levels of the HIV co-receptor CXCR4, induces the release of SDF-1 and inhibits HIV replication in Jurkat CD4+ T cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Jurkat cell lines stably expressing NS5A protein and peptides were generated and HIV replication in these cell lines assessed. HIV replication was significantly inhibited in all cell lines expressing NS5A amino acids 152-165. Substitution of an either alanine or glycine for the serine at position 158 (S158A or S158G) resulted in a significant decrease in the HIV inhibitory effect. In contrast, substituting a phosphomimetic amino acid (glutamic acid; S158E) inhibited HIV as well as the parent peptide. HIV inhibition was associated with lower levels of surface expression of the HIV co-receptor CXCR4 and increased release of the CXCR4 ligand, SDF-1 compared to control cells. Incubation of CD4+ T cell lines with synthetic peptides containing amino acids 152-167 or the S158E mutant peptide prior to HIV infection resulted in HIV replication inhibition compared to control peptides. CONCLUSIONS/SIGNIFICANCE: Expression of GBV-C NS5A amino acids 152-165 are sufficient to inhibit HIV replication in vitro, and the serine at position 158 appears important for this effect through either phosphorylation or structural changes in this peptide. The addition of synthetic peptides containing 152-167 or the S158E substitution to Jurkat cells resulted in HIV replication inhibition in vitro. These data suggest that GBV-C peptides or a peptide mimetic may offer a novel, cellular-based approach to antiretroviral therapy.

Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus.

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 alpha-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.

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus.

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.