NS5A Protein Research Articles

Electrical Immunosensor Made with Antigenic Peptide NS5A‑1 Immobilized onto Silk Fibroin for Diagnosing Hepatitis C

Immunosensors based on impedance spectroscopy for diagnosing hepatitis C are reported where the sensing units were made with the antigenic peptide PPLLESWKDPDYVPPWHG (NS5A-1) derived from the NS5A protein of the hepatitis C virus (HCV) immobilized in layer-by-layer (LbL) films with silk fibroin (SF) and deposited on gold interdigitated electrodes. The electrical response of the sensing units varied upon immersion into solutions containing the antibody anti-HCV owing to the biomolecular recognition of NS5A-1. This was associated with morphological changes on the LbL films caused by adsorption of NS5A-1 and inferred from atomic force microscopy images. Buffer solutions with different anti-HCV concentrations down to 2 ng mL-1 could be clearly distinguished by analyzing the impedance spectroscopy data with a multidimensional projection technique. The specificity toward anti-HCV antibodies was confirmed in control experiments where no significant changes in the electrical response were measured by exposing the sensing units to solutions containing an anti-human immunodeficiency virus (HIV) antibody. The high sensitivity and selectivity of the units made with LbL films demonstrate the feasibility of measuring electrical impedance as an immunosensing strategy to detect hepatitis C.

Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants

Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1-7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment. We measured the efficacy of different concentrations of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1-7 NS5A proteins with orwithout RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or thatcontained RAS previously reported from patients. NS5A inhibitors had varying levels of efficacy against original andresistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had high levels of fitness. Pibrentasvir had the highest level of efficacy against variants;viruses with RAS at amino acids 28, 30, or 31 hadnoapparent resistance to pibrentasvir, and HCV with RASat amino acid 93 had a low level of resistance to this drug.However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. Forthe remaining NS5A inhibitors tested, RAS atamino acids28 and 93 led to high levels of resistance. Among theseinhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants withRAS at amino acid 31 than the other agents. Variants withthepre-existing RAS T/Y93H acquired additional NS5Achanges during escape experiments, resulting in HCV variantswith specific combinations of RAS, showing high fitness and high resistance. We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent. These findings could improve treatment of patients with HCV infection.

Analysis of Naturally Occurring Resistance-Associated Variants to NS3/4A Protein Inhibitors, NS5A Protein Inhibitors, and NS5B Polymerase Inhibitors in Patients With Chronic Hepatitis C.

The first NS3/4A hepatitis C virus (HCV) protease inhibitors telaprevir and boceprevir were approved in 2011, and both NS5A and NS5B polymerase inhibitors were launched. Recently, direct-acting antivirals (DAAs) have had a major impact on patients infected with HCV. HCV DAAs are highly effective antivirals with fewer side effects. DAAs have been developed for the treatment of HCV infection in combination with PEG-IFN-α/RBV as well as in IFN-free regimens. However, some drug resistance mutations occur when a single oral DAA is used for treatment, which indicates that there is a low-frequency drug resistance mutation in HCV patients before the application of antiviral drugs. Our research showed that natural resistance to HCV DAAs was found in treatment-naive CHC patients and that the drug resistance mutation rates differ in various HCV genotypes. Many challenges posed by natural resistance should be considered in the context of DAA therapies.

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag “pull down” assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus.

Rab1A is required for assembly of classical swine fever virus particle

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein.

The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling

Flaviviruses are enveloped, positive-sensed single-stranded RNA viruses that remodel host membranes, incorporating both viral and host factors facilitating viral replication. In this study, we identified a key role for the membrane-bending host protein Reticulon 3.1 (RTN3.1A) during the replication cycle of three flaviviruses: West Nile virus (WNV), Dengue virus (DENV), and Zika virus (ZIKV). We observed that, during infection, RTN3.1A is redistributed and recruited to the viral replication complex, a recruitment facilitated via the WNV NS4A protein, however, not DENV or ZIKV NS4A. Critically, small interfering RNA (siRNA)-mediated knockdown of RTN3.1A expression attenuated WNV, DENV, and ZIKV replication and severely affected the stability and abundance of the NS4A protein, coinciding with a significant alternation and reduction of viral membrane structures in the endoplasmic reticulum. These observations identified a crucial role of RTN3.1A for the viral remodelling of host membranes during efficient flavivirus replication and the stabilization of viral proteins within the endoplasmic reticulum.

Prediction of drug-binding sites in HCV NS5B polymerase: MD simulation study

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense ribonucleic acid (RNA) virus and is a member of the Flaviviridae family of viruses. Its genome has a length of 9.6 kb and encoding a polyprotein of about 3000 amino acids which is cleaved co- and post-translationally by cellular and viral proteases into 10 different products: core, envelope protein E1, E2, p7, nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Core, E1 and E2 are the main viral constituents of the HCV particle. NS5B of the hepatitis C virus, a RNA-dependent RNA polymerase, is an important therapeutic target. In order to gain deeper insight into and to know its molecular mechanism, the authors employed molecular dynamics simulation at the nanosecond scale. Initial coordinates were taken from the protein data bank entries with IDs 3IGV and 3GYN. The present study shows the strong binding affinity of the inhibitor with NS5B polymerase. The binding free energies, calculated by the molecular mechanics and Poisson–Boltzmann surface area/generalized Born surface area method, suggest a good connection with the experimental biological activity. The analysis of the combined simulation results has identified that Tyr448, Ser556 and Asp318 are the key amino acid residues in NS5B binding. The results obtained from this study provide some insights into the development of novel strong NS5B inhibitors.

Secondary structure and membrane topology of dengue virus NS4A protein in micelles

Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication.

PCR Array Profiling of Antiviral Genes in Human Embryonic Kidney Cells Expressing Human Coronavirus OC43 Structural and Accessory Proteins

BackgroundHuman coronavirus OC43 (HCoV-OC43) causes common cold, and is associated with severe respiratory symptoms in infants, elderly and immunocompromised patients. HCoV-OC43 is a member of Betacoronavirus genus that includes also the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS) coronaviruses. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The objective of this study was to investigate the role of HCoV-OC43 structural (membrane and nucleocapsid) and accessory (ns5a and ns2a) proteins in the modulation of antiviral gene expression profile in human embryonic kidney 293 (HEK-293) cells using PCR array analysis.MethodsHCoV-OC43 membrane (M), nucleocapsid (N), ns5a and ns2a mRNA were amplified and cloned into the pAcGFP1-N expression vector (Clontech), followed by transfection in HEK-293 cells. Expression of M, N, ns5a and ns2a proteins were confirmed by indirect immunofluorescence test. Three days post-transfection, the cells were challenged by Sendai virus. The Human Antiviral Response PCR array system (Qiagen) was used to profile the antiviral gene expression in HEK-293 cells, using the fold regulation comparison and the manual normalisation methods.ResultsAround 50–60 genes were downregulated by HCoV-OC43 proteins, the most prominent genes being those critical for the activation of transcription factors involved in the antiviral response like interferon regulatory factors (IRFs) and activator protein 1 (AP-1). Among the most important downregulated genes were those coding for Interferons (IFNs) mitogen-activated protein kinases (MAPKs), pro-apoptotic and pyroptotic proteins (Caspases, cathepsins, tumour necrosis factor), pro-inflammatory cytokines (Interleukins), pattern recognition receptors (PRRs; toll-like receptors and NOD-like receptors) and their signaling transduction proteins (TICAM1, MAVS).ConclusionThis study shows for the first time that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways.Disclosures All authors: No reported disclosures.

Protein Inhibitor of Activated STAT2 Restricts HCV Replication by Modulating Viral Proteins Degradation.

Hepatitis C virus (HCV) replication in cells is controlled by many host factors. In this report, we found that protein inhibitor of activated STAT2 (PIAS2), which is a small ubiquitin-like modifier (SUMO) E3 ligase, restricted HCV replication. During infection, HCV core, NS3 and NS5A protein expression, as well as the viral assembly and budding efficiency were enhanced when endogenous PIAS2 was knocked down, whereas exogenous PIAS2 expression decreased HCV core, NS3, and NS5A protein expression and the viral assembly and budding efficiency. PIAS2 did not influence the viral entry, RNA replication, and protein translation steps of the viral life cycle. When expressed together with SUMO1, PIAS2 reduced the HCV core, NS3 and NS5A protein levels expressed from individual plasmids through the proteasome pathway in a ubiquitin-independent manner; the stability of these proteins in the HCV infectious system was enhanced when PIAS2 was knocked down. Furthermore, we found that the core was SUMOylated at amino acid K78, and PIAS2 enhanced the SUMOylation level of the core.

Safety and efficacy of sofosbuvir plus velpatasvir with or without ribavirin for chronic hepatitis C virus infection: A systematic review and meta-analysis.

Velpatasvir is a newly FDA-approved inhibitor of hepatitis C virus (HCV) NS5A protein. We performed this systematic review and meta-analysis to investigate the safety and efficacy of velpatasvir plus sofosbuvir in the treatment of chronic HCV infection. A computerized literature search of PubMed, SCOPUS, EMBASE, EBSCO, Web of science, and Cochrane CENTRAL was conducted using relevant keywords. Data from eligible studies were pooled in a fixed effect meta-analysis model, using OpenMeta[Analyst] software. Pooled data from six randomized trials (n=1427 patients) showed that velpatasvir plus sofosbuvir achieved sustained virological response (SVR12) rates of 98.2% in genotype-1, 99.4% in genotype-2, 94.7% in genotype-3, 99.6% in genotype-4, 97.1% in genotype-5, and 98.8% in genotype-6 patients. The addition of ribavirin did not significantly increase the SVR12 (RR=0.95, 95%CI [0.88, 1.02]) or decrease relapse rates (RR=2.52, 95% CI [0.49, 12.87]) in HCV genotype-1 patients. However, adding ribavirin significantly increased SVR12 (RR=89.5, 95% CI [80.4, 99.5]) in genotype-3 patients. In conclusion, the 12-week regimen of sofosbuvir plus velpatasvir was highly effective in HCV patients, including those with cirrhosis and former treatment experience. Except for genotype-3, adding ribavirin was not associated with significant improvements in SVR12 rates. Further studies should investigate the effect of adding ribavirin to this regimen, especially in HCV genotype-3 patients.

New Tethered Phospholipid Bilayers Integrating Functional G-Protein-Coupled Receptor Membrane Proteins.

Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10-7 cm2/s, and atomic force microscopy (AFM). By using variable lipid compositions including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol 4,5-bisphosphate (PIP2), sphingomyelin (SM), phosphatidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by the fusion of tethered proteoliposomes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2 × 10-7 cm2/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.

Hepatitis C virus down-regulates SERPINE1/PAI-1 expression to facilitate its replication.

Identification of host factors involved in viral replication is critical for understanding the molecular mechanism of viral replication and pathogenesis. Genes differentially expressed in HuH-7 cells with or without a hepatitis C virus (HCV) sub-genomic replicon were screened by microarray analysis. SERPINE1/PAI-1 was found to be down-regulated after HCV infection in this analysis. Down-regulation of SERPINE1/PAI-1 expression at the transcriptional level was verified by the real-time reverse transcriptase (RT)-PCR assay. Reduced SERPINE1/PAI-1 protein secretion was detected in the supernatant of HCV replicon cells and in sera from HCV-infected patients. SERPINE1 gene expression was down-regulated by HCV NS3/4A and NS5A proteins through the transforming growth factor-β (TGF-β) signalling pathway at the transcriptional level. Down-regulated genes in HCV replicon cells could be the factors supressing HCV replication. Indeed, over-expressed PAI-1 inhibited HCV replication but the mechanism is unknown. It has been demonstrated that HCV induces the expression of TGF-β, and TGF-β enhances HCV replication by a not-yet-defined mechanism. SERPINE1/PAI-1 is also known to be potently induced by TGF-β at the transcriptional level through both Smad-dependent and Smad-independent pathways. The exogenously expressed SERPINE1/PAI-1 suppressed the expression of the endogenous SERPINE1 gene at the transcriptional level through the TGF-β signalling but not the Smad pathway. Thus, SERPINE1/PAI-1 could suppress HCV replication possibly by negatively regulating TGF-β signalling. A model is proposed for the interplay betweenthe TGF-β signalling pathway, HCV and SERPINE1/PAI-1 to keep the homeostasis of the cells.

Hepatitis C Virus NS5A Targets Nucleosome Assembly Protein NAP1L1 To Control the Innate Cellular Response.

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A.IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition.

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein–phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH–PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH–PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein–phosphoinositide interactions.

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex.

Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

Zika Virus Hijacks Stress Granule Proteins and Modulates the Host Stress Response.

Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

Unconjugated interferon-stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I.

Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.

Spontaneous membrane insertion of a dengue virus NS2A peptide

Non-structural NS2A protein of Dengue virus is essential for viral replication but poorly characterized because of its high hydrophobicity. We have previously shown experimentally that NS2A possess a segment, peptide dens25, known to insert into membranes and interact specifically with negatively-charged phospholipids. To characterize its membrane interaction we have used two types of molecular dynamics membrane model systems, a highly mobile membrane mimetic (HMMM) and an endoplasmic reticulum (ER) membrane-like model. Using the HMMM system, we have been able of demonstrating the spontaneous binding of dens25 to the negatively-charged phospholipid 1,2-divaleryl-sn-glycero-3-phosphate containing membrane whereas no binding was observed for the membrane containing the zwitterionic one 1,2-divaleryl-sn-glycero-3-phosphocholine. Using the ER-like membrane model system, we demonstrate the spontaneous insertion of dens25 into the middle of the membrane, it maintained its three-dimensional structure and presented a nearly parallel orientation with respect to the membrane surface. Both charged and hydrophobic amino acids, presenting an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment, are responsible for membrane binding and insertion. Dens25 might control protein/membrane interaction and be involved in membrane rearrangements critical for the viral cycle. These data should help us in the development of inhibitor molecules that target NS2A segments involved in membrane reorganisation.