Hepatitis C Virus Nonstructural Protein 5A Inhibits Thapsigargin-Induced Apoptosis

Background

We previously reported that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) down-regulates TLR4 signaling and lipopolysaccharide-induced apoptosis of hepatocytes. There have been several reports regarding the association between HCV infection and endoplasmic reticulum (ER) stress. Here, we examined the regulation of HCV NS5A on the apoptosis of hepatocytes induced by thapsigargin, an inducer of ER stress.

Methods

The apoptotic response to thapsigargin and the expression of molecules involved in human hepatocyte apoptotic pathways were examined in the presence or absence of HCV NS5A expression.

Results

HCV JFH1 infection induced ER stress in the Huh7 cell line. HCV NS5A protected HepG2 cells against thapsigargin-induced apoptosis, the effect of which was linked to the enhanced expression of the 78-kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein (GRP78). Consistent with a conferred pro-survival advantage, HCV NS5A reduced poly(adenosine diphosphate-ribose) polymerase cleavage and activation of caspases-3, -7 and -9, and Bax expression, while increasing the expressions of the anti-apoptotic molecules XIAP and c-FLIP. HCV NS5A weakly interacts with GRP78 and enhances GRP78 expression in hepatocytes.

Conclusion

HCV NS5A enhances GRP78 expression, resulting in the inhibition of apoptotic properties, and inhibits thapsigargin-induced apoptotic pathways in human hepatocytes, suggesting that disruption of ER stress-mediated apoptosis may have a role in the pathogenesis of HCV infection. Thus, HCV NS5A might engender the survival of HCV-infected hepatocytes contributing to the establishment of persistent infection.     

Toscana Virus NSs Protein Inhibits the Induction of Type I Interferon by Interacting with RIG-I

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71–79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.     

Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.     

Foot-and-Mouth Disease Virus Nonstructural Protein 2C Interacts with Beclin1, Modulating Virus Replication

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.     

Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG^Met→GCC^Ala mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.     

Intracellular Distribution of Rubella Virus Nonstructural Protein P150

Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells. Replicase protein P150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. Newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral RNA synthesis.     

流感病毒A非结构蛋白功能的研究进展

流感病毒属正粘病毒科(Orthomyxoviridae),是一种带包膜并且分节段的单负链RNA病毒。根据病毒核衣壳蛋白(Nucleocapsid)和基质蛋白(Matrix,M)抗原性的差异,流感病毒可分为甲、乙、丙3个型。甲型流感病毒呈球形或丝状,其RNA基因组总长度在13.6kb左右,分为8个节段,共编码10个蛋白     

Localization of the Single-Stranded RNA-Binding Domains of Bluetongue Virus Nonstructural Protein NS2

The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.     

基孔肯雅病毒囊膜蛋白假病毒模型的构建

目的:以HIV为骨架构建单次复制性的含基孔肯雅病毒囊膜蛋白的假病毒模型,观察其对哺乳动物细胞的侵染性。方法:PCR合成基孔肯雅病毒囊膜蛋白基因,克隆到真核表达载体上,与HIV慢病毒包装系统质粒共转染293FT细胞,48 h后收培养上清,在8μg/mL Polybrene存在下感染293FT细胞,感染48 h后在荧光显微镜下观察结果。结果:PCR合成了基孔肯雅病毒囊膜蛋白基因并克隆到真核表达载体上,测序结果正确;共转染293FT细胞后,检测到基孔肯雅病毒囊膜蛋白的表达并包装成假病毒,感染新鲜293FT细胞后能够检测到绿色荧光蛋白。结论:合成的基孔肯雅病毒囊膜蛋白基因能正确表达并包装成假病毒,含基孔肯雅病毒囊膜蛋白的假病毒能感染293FT细胞并表达绿色荧光蛋白,可用该假病毒模型进一步研究基孔肯雅病毒的感染性,筛选评价抗基孔肯雅病毒药物。     

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.An estimated 130 million people are infected with hepatitis C virus (HCV), the etiologic agent of non-A, non-B viral hepatitis. Transmission of the virus occurs primarily through blood or blood products. Acute infections are frequently asymptomatic, and 70 to 80% of the infected individuals are unable to eliminate the virus. Of the patients with HCV-induced chronic hepatitis, 15 to 30% progress to cirrhosis within years to decades after infection, and 3 to 4% of patients develop hepatocellular carcinoma (17). HCV infection is a leading cause of cirrhosis, end-stage liver disease, and liver transplantation in Europe and the United States (7), and reinfection after liver transplantation occurs almost universally. There is no vaccine available, and current HCV therapy of pegylated alpha interferon in combination with ribavirin leads to a sustained response in only about 50% of genotype 1-infected patients.The positive-stranded RNA genome of HCV is about 9.6 kb in length and encodes a single open reading frame flanked by 5′ and 3′ nontranslated regions (5′ and 3′ NTRs). The translation product of the viral genome is a large polyprotein containing the structural proteins (core, envelope proteins E1 and E2) in the N-terminal region and the nonstructural proteins (p7, nonstructural protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B) in the C-terminal region. The individual proteins are processed from the polyprotein by various proteases. The host cellular signal peptidase cleaves between core/E1, E1/E2, E2/p7, and p7/NS2, and signal peptide peptidase releases core from the E1 signal peptide. Two viral proteases, the NS2-3 protease and the NS3-4A protease, cleave the remainder of the viral polyprotein in the nonstructural region (22, 27). The structural proteins package the genome into infectious particles and mediate virus entry into a naïve host cell; the nonstructural proteins NS3 through NS5B form the RNA replication complex. p7 and NS2 are not thought to be incorporated into the virion but are essential for the assembly of infectious particles (14, 36); however, their mechanisms of action are not understood.NS2 (molecular mass of 23 kDa) is a hydrophobic protein containing several transmembrane segments in the N-terminal region (5, 9, 32, 39). The C-terminal half of NS2 and the N-terminal third of NS3 form the NS2-3 protease (10, 11, 26, 37). NS2 is not required for the replication of subgenomic replicons, which span NS3 to NS5B (20). However, cleavage at the NS2/3 junction is necessary for replication in chimpanzees (16), the full-length replicon (38), and in the infectious tissue culture system (HCVcc) (14). Although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at the NS2/3 site (32). In vitro studies indicate that purified NS2-3 protease is active in the absence of cellular cofactors (11, 37). In addition to its role as a protease, NS2 has been shown to be required for assembly of infectious intracellular virus (14). The N-terminal helix of NS2 was first implicated in infectivity by the observation that an intergenotypic breakpoint following this transmembrane segment resulted in higher titers of infectious virus (28). Structural and functional characterization of the NS2 transmembrane region has shown that this domain is essential for infectious virus production (13). In particular, a central glycine residue in the first NS2 helix plays a critical role in HCV infectious virus assembly (13). The NS2 protease domain, but not its catalytic activity, is also essential for infectious virus assembly, whereas the unprocessed NS2-3 precursor is not required (13, 14).The crystal structure of the postcleavage NS2 protease domain (NS2^pro, residues 94 to 217), revealed a dimeric cysteine protease containing two composite active sites (Fig. 2C; [21]). Two antiparallel α-helices make up the N-terminal subdomain, followed by an extended crossover region, which positions the β-sheet-rich C-terminal subdomain near the N-terminal region of the partner monomer. Two of the conserved residues of the catalytic triad (His 143, Glu 163) are located in the loop region after the second N-terminal helix of one monomer, while the third catalytic residue, Cys 184, is located in the C-terminal subdomain of the other monomer. Creation of this unusual pair of composite active sites through NS2 dimerization has been shown to be essential for autoproteolytic cleavage (21). The structure of NS2^pro further demonstrated that the C-terminal residue of NS2 remains bound in the active site after cleavage, suggesting a possible mechanism for restriction of this enzyme to a single proteolytic event (21). Here we have used the crystal structure of NS2^pro, along with sequence alignments, to target conserved residues in each of the NS2^pro structural regions. Our mutational analysis revealed that the residues in the dimer crossover region and the C-terminal subdomain are important for infectious virus production. In contrast, the majority of amino acids in the active site pocket were not required for infectivity. Interestingly, we observed that the extreme C-terminal leucine of NS2 is absolutely essential for generation of infectious virus, as mutations, deletions, and extensions into NS3 are very poorly tolerated. This analysis begins to dissect the determinants of the multiple functions of this important protease in the HCV life cycle.