Full-length NS3 Research Articles

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Norovirus MLKL-like protein initiates cell deathto induce viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2′-methyl-2′-fluoro guanosine 5′-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2′-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2′-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

A mechanism for transcriptional regulation of innate immune response genes by RSV NS1 protein.

Human Respiratory syncytial virus (RSV) is a main cause of bronchiolitis in young children and a still unmet target for pediatric vaccines and antiviral drugs. RSV has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2, identified as interferon antagonists. Recently it was suggested that NS1 modulates host gene transcription via interactions with chromatin and the Mediator complex. Moreover, the C-terminal RSV NS1 α-helix, α3, was reported to be involved in modulation of host response and gene expression. Here, after identifying the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen, we demonstrated that NS1 directly interacts with MED25 in cells and in vitro. We assigned this interaction to the MED25-ACID domain, a target of viral transcriptional activators, such as Herpes simplex virus VP16. We showed that NS1 α3 primarily targets the VP16 transactivation domain (TAD) binding site of MED25-ACID. In particular, we found out that NS1 could compete with the TAD of ATF6α, a master regulator of ER stress response activated upon viral infection, for MED25-ACID binding. The affinity for MED25-ACID determined using an NS1-α3 peptide was surprisingly low, as compared to that of full-length NS1. Although this can be partly explained by the unfolded state of the isolated peptide, it suggests the presence of a second binding site. We indeed observed that the NS1Δα3 deletion mutant was able to bind to MED25-ACID. Altogether our findings suggest a mechanism for transcription modulation of specific genes by RSV NS1, and hence for modulation of the host immune response, where NS1 competes with cellular TADs for recruitment of the Mediator MED25 subunit.

A variant NS1 protein from H5N2 avian influenza virus suppresses PKR activation and promotes replication and virulence in mammals

ABSTRACT Highly pathogenic avian influenza viruses (HPAIVs) frequently receive global attention as threats to public health. The NS1 protein is a key virulence factor known to impair host antiviral responses. The study herein revealed HPAIV H5N2 NS gene encoded additional protein; a truncated NS1 variant, designated NS3, produced by alternative splicing of the NS transcript. To examine the function of NS3 during infection, we generated recombinant viruses expressing either full-length NS1 (RG-AIV-T375G) or NS3 (RG-AIV-NS3). Interestingly, RG-AIV-NS3 virus produced higher titres than RG-AIV-T375G in multiple mammalian cell lines. However, RG-AIV-T375G exhibited a replication advantage over RG-AIV-NS3 in chicken DF-1 cells, indicating that host cell identity dictates the effect of NS3 on viral replication. In mice and mammalian cells, RG-AIV-NS3 infection elicited higher level of cytokines, including IFN-β, MX and TNF-α, potentially due to its higher replication activity. Based on mini-genome assay, NS3 had pronounced effects on viral replication machinery. Surprisingly, NS3 retained an interaction with PKR and suppressed PKR activation despite its lack of amino-acid residues 126-167. The poor replication ability of RG-AIV-T375G was partially restored in cells deficient in PKR suggesting that full-length NS1 may be insufficient to suppress PKR function. Notably, virulence of the full-length NS1-expressing RG-AIV-T375G virus was highly attenuated in mice when compared to RG-AIV-NS3. In summary, our study reveals the existence and function of a previously unidentified H5N2 viral protein, NS3. We found that NS3 is functionally distinct from NS1 protein, as it enhances viral replication and pathogenicity in mammalian systems, potentially via suppression of PKR activity.

Hesperidin identified from Citrus extracts potently inhibits HCV genotype 3a NS3 protease

BackgroundHepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries.MethodsThe full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI–MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease.ResultsFusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MSn analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC50 value of 11.34 ± 3.83 µg/mL.ConclusionsA FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.

Influenza A Virus NS1 Protein Structural Flexibility Analysis According to Its Structural Polymorphism Using Computational Approaches

Influenza A viruses are highly contagious RNA viruses that cause respiratory tract infections in humans and animals. Their non-structural protein NS1, a homodimer of two 230-residue chains, is the main viral factor in counteracting the antiviral defenses of the host cell. Its RNA-binding domain is an obligate dimer that is connected to each of the two effector domains by a highly flexible unstructured linker region of ten amino acids. The flexibility of NS1 is a key property that allows its effector domains and its RNA binding domain to interact with several protein partners or RNAs. The three-dimensional structures of full-length NS1 dimers revealed that the effector domains could adopt three distinct conformations as regards their mutual interactions and their orientation relative to the RNA binding domain (closed, semi-open and open). The origin of this structural polymorphism is currently being investigated and several hypotheses are proposed, among which one posits that it is a strain-specific property. In the present study, we explored through computational molecular modeling the dynamic and flexibility properties of NS1 from three important influenza virus A strains belonging to three distinct subtypes (H1N1, H6N6, H5N1), for which at least one conformation is available in the Protein Data Bank. In order to verify whether NS1 is stable in three forms for the three strains, we constructed homology models if the corresponding forms were not available in the Protein Data Bank. Molecular dynamics simulations were performed in order to predict the stability over time of the three distinct sequence variants of NS1, in each of their three distinct conformations. Our results favor the co-existence of three stable structural forms, regardless of the strain, but also suggest that the length of the linker, along with the presence of specific amino acids, modulate the dynamic properties and the flexibility of NS1.

Alignment of helicases on single-stranded DNA increases activity.

Helicases function in most biological processes that utilize RNA or DNA nucleic acids including replication, recombination, repair, transcription, splicing, and translation. They are motor proteins that bind ATP and then catalyze hydrolysis to release energy which is transduced for conformational changes. Different conformations correspond to different steps in a process that results in movement of the enzyme along the nucleic acid track in a unidirectional manner. Some helicases such as DEAD-box helicases do not translocate, but these enzymes transduce chemical energy from ATP hydrolysis to unwind secondary structure in DNA or RNA. Some helicases function as monomers while others assemble into defined structures, either dimers or higher order oligomers. Dda helicase from bacteriophage T4 and NS3 helicase domain from the hepatitis C virus are examples of monomeric helicases. These helicases can bind to single-stranded DNA in a manner that appears like train engines on a track. When monomeric helicases align on DNA, the activity of the enzymes increases. Helicase activity can include the rate of duplex unwinding and the total number of base pairs melted during a single binding event or processivity. Dda and NS3h are considered as having low processivity, unwinding fewer than 50 base pairs per binding event. Here, we report fusing two molecules of NS3h molecules together through genetically linking the C-terminus of one molecule to the N-terminus of a second NS3h molecule. We observed increased processivity relative to NS3h possibly arising from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. The dimeric enzyme also binds DNA more like the full-length NS3 helicase. Finally, the dimer can displace streptavidin from biotin-labeled oligonucleotide, whereas monomeric NS3h cannot.

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains.
 MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs).
 RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals.
 CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Purification of Dengue and Zika Virus Non-structural Protein 5 for Crystallization and Screening of Antivirals.

Dengue Virus (DENV) and ZIKA Virus (ZIKV) are two important human pathogens that belong to the Flavivirus genus of positive strand RNA viruses. Symptoms of DENV infections range from asymptomatic or mild fever to life-threatening forms, while ZIKV can lead to teratogenic effects such as microcephaly in newborns and neurological disease like the Guillain-Barré syndrome.Non-Structural Protein 5 (NS5) is the largest and most conserved enzyme across flaviviruses and hence constitutes a prime target for developing pan-flavivirus antiviral inhibitors. NS5 results from the gene fusion between a methyltransferase at the N-terminus of the protein and an RNA-dependent RNA polymerase (RdRp) at the C-terminal end. The NS5 protein plays key roles in replication and modification of viral RNA and its inhibition by potent antiviral drugs could prevent severe symptoms associated with infections.We have optimized purification and crystallization protocols to obtain active recombinant proteins suitable for structure-based drug discovery for both the full-length NS5 protein and the polymerase domain of NS5 from DENV and ZIKV .

Potent novel inhibitors against hepatitis C virus NS3 (HCV NS3 GT-3a) protease domain

HCV NS3, a non-structural hepatitis C viral protein is used as one of the potential targets for inhibition by direct-acting antivirals. It is known that the success rate for HCV genotype-1 treatment remained very high, however, treatment of genotype-3a (GT-3a), is still quite challenging. In the current study, the HCV GT-3a full-length NS3 gene was amplified and sequenced. The complete nucleotide sequence was translated into the amino acid sequence and homology models of HCV-NS3 GT-3a were generated by HCV-NS3 genotype-1b as a template. The objective of the study was to screen novel therapeutic hits from large databases. For this aim, various small molecule databases including, BindingDB (∼45.000 compounds), NCI (∼265.000 compounds), and Specs-SC (∼212.000 compounds) were used. Firstly, all of the compounds were screened using binary-QSAR models from the MetaCore/MetaDrug server, and compounds were filtered based on therapeutic activity predictions by the anti-viral QSAR model. Filtered molecules were used in 26 different toxicity QSAR models and active non-toxic compounds were identified. These selected molecules were then used in docking and molecular dynamics (MD) simulations studies at the binding cavities of the NS3 protease domain of the GT-3a. Results were compared with known inhibitors and novel molecules are proposed against HCV-NS3 GT-3a. These molecules have high ligand efficiencies as compared to the reference molecules suggesting a better alternate to the existing suite of inhibitors. Thus, this study will be a step ahead in the development of new potential compounds as antiviral drugs for the GT-3a target.

Enhanced CD8+ T-cell response in mice immunized with NS1-truncated influenza virus

Influenza viruses with truncated NS1 protein stimulate a more intensive innate immune response compared to their wild type counterparts. Here, we investigate how the shortening of the NS1 protein influence the immunogenicity of the conserved T-cellular epitopes of influenza virus. Using flow cytometry, we showed that the intraperitoneal immunization of mice with influenza virus encoding 124 N-terminal amino acid residues of the NS1 protein (A/PR8/NS124) induced higher levels of CD8+ T-cells recognizing immunodominant (NP366-374) and sub-immunodominant (NP161-175, NP196-210, HA323-337, HA474-483, NA427-433) epitopes compared to immunization with the virus expressing full-length NS1 (A/PR8/full NS). It is noteworthy that the response to the immunodominant influenza epitope NP366-374 was achieved with the lower immunization dose of A/PR8/NS124 virus compared to the reference wild type strain. Despite the fact that polyfunctional CD8+ effector memory T-lymphocytes simultaneously producing two (IFNγ and TNFα) or three (IFNγ, IL2, and TNFα) cytokines prevailed in the immune response to both viruses, the relative number of such T-cells was higher in A/PR8/NS124-immunized mice. Furthermore, we have found that polyfunctional populations of lymphocytes generated upon the immunization of mice with the mutant virus demonstrated an increased capacity to produce IFNγ compared to the corresponding populations derived from the A/PR8/full NS-immunized mice. Therefore, immunization with the attenuated influenza virus encoding truncated NS1 protein ensures a more potent CD8+ T-cell immune response.

Advance in research on structure and functions of the nonstructural protein 5 of Zika virus

Zika virus (ZIKV) is an important member of genus Flavivirus which includes dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. ZIKV has attracted worldwide attention because of its unexpected causal link to microcephaly, Guillain-Barre syndrome, and other serious neurological diseases. So far, it has been spreading to 90 countries or territories worldwide, and approximately 3.6 billion people are now living at risk of ZIKV infection. Although a lot of efforts have been made to understand the pathogenesis of ZIKV, there is no approved vaccines or antiviral drugs available. ZIKV has a single-stranded and positive-sense genome RNA, which contains a single open reading frame flanked by the highly structured untranslated regions (UTRs). The ORF encodes a polyprotein precursor which is further cleaved into 3 structural proteins (C, prM/M and E) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by either host or viral protease. ZIKV NS5 is the largest and the most conserved protein among all viral proteins. ZIKV NS5 can be divided into two domains: the N-terminal methyltransferase (MTase) domain and C-terminal RNA-dependent RNA polymerase (RdRp) domain. Recently, the high-resolution structure of full-length ZIKV NS5 protein as well as the individual MTase and RdRp domains have been resolved, which led to better understanding of its biological function. As a multifunctional protein, ZIKV NS5 interacts with several cis -acting RNA elements located in the 5′UTR to initiate viral RNA synthesis, and adds the 5′RNA cap structure to facilitate translation of the polyprotein. NS5 is also involved in the regulation of host innate antiviral immune responses. As such, ZIKV NS5 is widely considered to be a potential target for antiviral drug design, and the research towards this goal also accelerated our understanding of its structure and function. A panel of small molecule inhibitors targeting ZIKV NS5 has been identified to have ideal antiviral effects in both cell cultures and animal models. This review summarizes recent advances on the structure and biological functions of ZIKV NS5 protein.

Screening of Duck Tembusu Virus NS3 Interacting Host Proteins and Identification of Its Specific Interplay Domains.

NS3 protein is a member of the non-structural protein of duck Tembusu virus (DTMUV), which contains three domains, each of which has serine protease, nucleotide triphosphatase, and RNA helicase activities, respectively. It performs a variety of biological functions that are involved in the regulation of the viral life cycle and host immune response. Based on the yeast two-hybrid system, we successfully transformed pGBKT7-NS3 bait plasmid into Y2H Gold, tested it to prove that it has no self-activation and toxicity, and then hybridized it with the prey yeast strain of the duck embryo fibroblast cDNA library for screening. After high-stringency selection, positive alignment with the National Center for Biotechnology Information database revealed nine potential interactive proteins: MGST1, ERCC4, WIF1, WDR75, ACBD3, PRDX1, RPS7, ND5, and LDHA. The most interesting one (PRDX1) was selected to be verified with full-length NS3 protein and its three domains S7/DEXDc/HELICc using yeast regressive verification and GST Pull-Down assay. It denoted that PRDX1 does indeed interact with HELICc domains of NS3. NS3 is involved in the RNA uncoiling process of viral replication, which may cause mitochondrial overload to create oxidative stress (OS) during DTMUV attack. We deduced that the HELICc domain binding partner PRDX1, which regulates the p38/mitogen-activated protein kinase pathway (p38/MAPK) to avert OS, causing apoptosis, making it possible for viruses to escape host immune responses.

Structure of the yellow fever NS5 protein reveals conserved drug targets shared among flaviviruses

Yellow fever virus (YFV) is responsible for devastating outbreaks of Yellow fever (YF) in humans and is associated with high mortality rates. Recent large epidemics and epizootics and exponential increases in the numbers of YF cases in humans and non-human primates highlight the increasing threat YFV poses, despite the availability of an effective YFV vaccine. YFV is the first human virus discovered, but the structures of several of the viral proteins remain poorly understood. Here we report the structure of the full-length NS5 protein, a key enzyme for the replication of flaviviruses that contains both a methyltransferase domain and an RNA dependent RNA polymerase domain, at 3.1 Å resolution. The viral polymerase adopts right-hand fold, demonstrating the similarities of the Yellow fever, Dengue and Zika polymerases. Together this data suggests NS5 as a prime and ideal target for the design of pan-flavivirus inhibitors.

Supramolecular arrangement of the full-length Zika virus NS5.

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.

Recombinant DENV 2 NS5: An effective antigen for diagnosis of DENV infection

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg2+-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg2+ or Mn2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Detection of a novel human pegivirus HPgV-2 in healthy blood donors and recipients of multiple transfusions: implications for blood safety

To investigate the prevalence of HPgV-2 in blood donors, transfusion recipients and hemophilia patients and its impact on blood safety. Serum samples were collected from 1060 healthy blood donors, 1402 HCV-positive and 500 HBV- positive blood donors, 570 transfusion recipients and 248 hemophilia patients for screening anti-HPgV-2 antibodies, HPgV-2 RNA, anti-HCV and HBsAg/HBV-DNA using ELISA and RT-PCR. Phylogenetic analysis of near fulllength genome sequences and NS3 genes of pegiviruses and hepaciviruses were performed using MEGA software. Anti-HPgV-2 positivity and HPgV-2 RNA positivity were found in 1.21% (17/1402) and 0.36% (5/1402) of the blood donors infected with HCV (RNA+/Ab+), respectively, indicating a close correlation between HPgV-2 and HCV infection (χ2=13.78, P= 0.004). Anti-HPgV-2 antibody was hardly detected in the other populations. A nucleotide identity as high as 97.11% was found in the NS3 fragments among the 5 isolated HPgV-2 strains, which had a nucleotide identity of 96.53% with the reported strains isolated out of China. The prevalence of HPgV-2 infection is rather low in healthy blood donors and transfusion recipients. Coinfection with HCV is common in HPgV-2 infection, and no evidence has now been available to support HPgV-2 transmission via blood transfusion, indicating that HPgV-2 may not pose a threat to blood safety.

Evaluation of Sofosbuvir (\u03b2-D-2\u2032-deoxy-2\u2032-\u03b1-fluoro-2\u2032-\u03b2-C-methyluridine) as an inhibitor of Dengue virus replication#

We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine-5′-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.