Virus Nonstructural Protein Research Articles

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways.

Hepatic fibrosis is a serious condition, and the development of hepatic fibrosis can lead to a series of complications. However, the pathogenesis of hepatic fibrosis remains unclear, and effective therapy options are still lacking. Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1 (NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis, but its role in diseases including hepatic fibrosis remains undefined. Therefore, additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment. To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1. Twenty-four male C57BL/6 mice were randomized and separated into three groups, comprising the normal, fibrosis, and calcitriol treatment groups, and liver fibrosis was modeled by carbon tetrachloride (CCl4). To evaluate the level of hepatic fibrosis in every group, serological and pathological examinations of the liver were conducted. TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells. NS3TP1, α-smooth muscle actin (α-SMA), collagen I, and collagen III in every group were examined using a Western blot and real-time quantitative polymerase chain reaction. The activity of the transforming growth factor beta 1 (TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected. The statistical analysis of the data was performed using the Student's t test. NS3TP1 promoted the activation, proliferation, and differentiation of hepatic stellate cells (HSCs) and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways, as evidenced by the presence of α-SMA, collagen I, collagen III, p-smad3, and p-p65 in LX-2 cells, which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference. The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression, as shown by the luciferase assay. NS3TP1 inhibited the apoptosis of HSCs. Moreover, both Smad3 and p65 could bind to NS3TP1, and p65 increased the promoter activity of NS3TP1, while NS3TP1 increased the promoter activity of TGFβ1 receptor I, as indicated by coimmunoprecipitation and luciferase assay results. Both in vivo and in vitro, treatment with calcitriol dramatically reduced the expression of NS3TP1. Calcitriol therapy-controlled HSCs activation, proliferation, and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice. Furthermore, calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1. Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique, prospective therapeutic target in hepatic fibrosis.

Prediction of human protein interactome of dengue virus non-structural protein 5 (NS5) and its downstream immunological implications.

The online version contains supplementary material available at 10.1007/s13205-023-03569-0.

Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.

HDAC6 Degrades nsp8 of Porcine Deltacoronavirus through Deacetylation and Ubiquitination to Inhibit Viral Replication.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.

Rab27-dependent extracellular vesicle releasing participates in dengue virus non-structural protein 1 secretion

Abstract Dengue disease is one of important arthropod-borne disease in the world and causes around 20,000 deaths annually. Dengue virus (DENV) non-structural protein 1 (NS1) is the major secreted non-structural protein from infected cells and highly associated with the pathogenesis of severe dengue. Several studies have shown that the secreted NS1 contribute to increase of vascular permeability, coagulopathy, activation of immune cells as well as inflammation. These findings strongly indicate the pathological roles of secreted NS1; however, the secretory pathway is not fully clear. Accumulating evidence have shown that virus-associated extracellular vesicles (EVs) not only contain viral components including viral proteins, genetic material or virion particle but also host components. Rab27 is one of several GTPases essential to regulate EV releasing by affecting docking of multivesicular body (MVB) to the target plasma membrane. Here, we show that NS1 can be exported from DENV-infected cells by EVs. NS1 as well as EV biogenesis-associated proteins ALG-2-interacting protein X (ALIX) and neutral sphingomyelinase2 (nSMase2) co-accumulate in extracellular vesicles. We next determine the topology of NS1 and demonstrate that NS1 is associated at the surface of EVs. In addition, the secretion of DENV NS1 is regulated by Rab27. The EV biogenesis and secretion inhibitor, Tipifarnib treatment decreases the levels of NS1 and nSMase 2 but not the levels of ALIX in the EVs. Taken together, this study not only provide the new insights of mechanisms in DENV NS1 secretion through Rab27-mediated vesicle trafficking but also valuable therapeutic targets to dengue disease. Supported by grants from Ministry of Science and Technology of Taiwan (MOST 111-2320-B-006 -051 -MY3)

Identification of Key Residues in Dengue Virus NS1 Protein That Are Essential for Its Secretion.

Dengue virus (DENV) non-structural protein 1 (NS1) is involved in multiple aspects of the DENV lifecycle. Importantly, it is secreted from infected cells as a hexameric lipoparticle that mediates vascular damage that is a hallmark of severe dengue. Although the secretion of NS1 is known to be important in DENV pathogenesis, the exact molecular features of NS1 that are required for its secretion from cells are not fully understood. In this study, we employed random point mutagenesis in the context of an NS1 expression vector encoding a C-terminal HiBiT luminescent peptide tag to identify residues within NS1 that are essential for its secretion. Using this approach, we identified 10 point mutations that corresponded with impaired NS1 secretion, with in silico analyses indicating that the majority of these mutations are located within the β-ladder domain. Additional studies on two of these mutants, V220D and A248V, revealed that they prevented viral RNA replication, while studies using a DENV NS1-NS5 viral polyprotein expression system demonstrated that these mutations resulted in a more reticular NS1 localisation pattern and failure to detect mature NS1 at its predicted molecular weight by Western blotting using a conformation-specific monoclonal antibody. Together, these studies demonstrate that the combination of a luminescent peptide tagged NS1 expression system with random point mutagenesis enables rapid identification of mutations that alter NS1 secretion. Two such mutations identified via this approach revealed residues that are essential for correct NS1 processing or maturation and viral RNA replication.

Perspectives on the current antiviral developments towards RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase) domains of dengue virus non-structural protein 5 (DENV-NS5)

Dengue virus (DENV) infection is one of the most emerging arboviral infections in humans. DENV is a positive-stranded RNA virus in the Flaviviridae family consisting of an 11 kb genome. DENV non-structural protein 5 (DENV-NS5) constitutes the largest among the non-structural proteins, which act as two domains, the RNA-dependent RNA polymerase (RdRp) and RNA methyltransferase enzyme (MTase). The DENV-NS5 RdRp domain contributes to the viral replication stages, whereas the MTase initiates viral RNA capping and facilitates polyprotein translation. Given the functions of both DENV-NS5 domains have made them an important druggable target. Possible therapeutic interventions and drug discoveries against DENV infection were thoroughly reviewed; however, a current update on the therapeutic strategies specific to DENV-NS5 or its active domains was not attempted. Since most potential compounds and drugs targeting the DENV-NS5 were evaluated in both in vitro cultures and animal models, a more detailed evaluation of molecules/drug candidates still requires investigation in randomized controlled clinical trials. This review summarizes current perspectives on the therapeutic strategies adopted to target the DENV-NS5 (RdRp and MTase domains) at the host-pathogen interface and further discusses the directions to identify candidate drugs to combat DENV infection.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

Stabilization of the Quadruplex-Forming G-Rich Sequences in the Rhinovirus Genome Inhibits Uncoating-Role of Na+ and K.

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na+ but not in K+-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na+ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published.

Risk mapping using serologic surveillance for selected One Health and transboundary diseases in Cambodian goats.

In Cambodia, goat production and meat consumption are customary among Muslim communities. Recently, goat meat has gained popularity among Cambodians. Goat farmers use a traditional management system, including grazing, requiring minimal labour. The close proximity between humans and animals could increase the risk of zoonotic disease transmission. A serological survey was undertaken to estimate the prevalence of some priority zoonoses and high-impact animal diseases in the Cambodian goat population. A total of 540 samples were collected from goats in six provinces and analysed with commercially available enzyme-linked immunosorbent assays for Brucella species, Q fever (Coxiella burnetii), Foot and Mouth Disease virus non-structural protein (FMDV NSP) and Peste des Petits Ruminants virus (PPRV). True seroprevalences with a 95% Confidence Interval (CI), taking into account imperfect tests, risk factors and odds ratios (ORs), were calculated to better understand the disease distribution and epidemiology. Independent variables used in statistical modellings included sex, body condition score, age, vaccination history, province and commune, while dependent variables were ELISA test results. The overall true prevalence of antibodies to Brucella spp., C. burnetii, FMDV and PPRV, were 0.1% (95% CI 0.0, 1.0), 7.2% (95% CI 5.3, 9.7), 57.7% (95% CI 53.1, 62.3) and 0.0% (95% CI 0.0, 0.0), respectively. There was no identified risk factor for brucellosis and PPR. The two risk factors for C. burnetii seropositivity were sex (p-value = 0.0005) and commune (p-value <0.0001). However, only the OR of C. burnetii seropositive female goat was significant at 9.7 (95% CI 2.7, 35.5) times higher than male. The risk factors of FMD NSP seropositivity were age (p-value = 0.001) and commune (p-value <0.0001). Only the age 'more than two-year-old' group with a significant OR of 6.2 (95% CI 2.1, 18.4) using the 'up to one-year-old' group as the reference. In summary, Brucella spp. seroprevalence was low, while no evidence of PPRV antibodies was detected in the goat populations. C. burnetii seroprevalence in female goats was significantly higher than for males, and there were significant differences in C. burnetii seroprevalence between communes. The overall FMDV NSP seroprevalence was high, especially in older animals. Vaccination should be advocated to protect animals from FMDV and improve productivity. As the impacts of these zoonoses on human and animal health were still unknown, further investigation of these zoonotic diseases' epidemiology is recommended.

Hepatitis C virus nonstructural protein 4B induces lipogenesis via the Hippo pathway

Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural protein 4B (NS4B) has been reported to induce lipogenesis, but the underlying mechanism is unclear. In this study, western blots were performed to investigate the effect of NS4B protein levels on key effectors of the Hippo and AKT signaling pathways. Yes-associated protein (YAP) and moesin-ezrin-radixin-like protein (Merlin) are effectors of the Hippo pathway. NS4B downregulated Merlin and phosphorylated YAP (p-YAP) protein expression while increasing the expression of the key AKT pathway proteins p-AKT and NF-κB. By observing the levels of AKT pathway proteins when Merlin was overexpressed or silenced, it was determined that Merlin mediates the AKT pathway. We suggest that HCV NS4B may mediate the AKT signaling pathway by inhibiting the Hippo pathway. Lipid droplets were observed in Huh7.5 cells overexpressing NS4B, and they increased significantly in number when Merlin was silenced. Overexpression of NS4B and Merlin silencing enhanced the expression of sterol regulatory element binding proteins (SREBPs), which have been demonstrated to be key regulatory factors controlling fatty acid synthesis. NS4B and Merlin silencing also enhanced the in vitro proliferative capacity of hepatocellular carcinoma cells. In conclusion, NS4B induces lipogenesis via the effect of the Hippo-YAP pathway on the AKT signaling pathway and thereby plays a significant role in the pathogenesis of HCV-associated diseases.

Development of Enterovirus Antiviral Agents That Target the Viral 2C Protein.

The Enterovirus (EV) genus includes several important human and animal pathogens. EV-A71, EV-D68, poliovirus (PV), and coxsackievirus (CV) outbreaks have affected millions worldwide, causing a range of upper respiratory, skin, and neuromuscular diseases, including acute flaccid myelitis, and hand-foot-and-mouth disease. There are no FDA-approved antiviral therapeutics for these enteroviruses. This study describes novel antiviral compounds targeting the conserved non-structural viral protein 2C with low micromolar to nanomolar IC50 values. The selection of resistant mutants resulted in amino acid substitutions in the viral capsid protein, implying these compounds may play a role in inhibiting the interaction of 2C and the capsid protein. The assembly and encapsidation stages of the viral life cycle still need to be fully understood, and the inhibitors reported here could be useful probes in understanding these processes.

Development of a novel real-time RT-PCR method using peptide nucleic acid (PNA) probes for detecting and genotyping of viral haemorrhagic septicaemia virus (VHSV)

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. The causative agent of VHS is the VHS virus (VHSV), which has been classified into four genotypes (I–IV), based on sequence analysis of the genes encoding for nucleoprotein, glycoprotein, and non-structural viral protein. Among the various diagnostic methods, real-time reverse transcription PCR method based on TaqMan-probe (RT-qPCR) is a stable, rapid, specific, and highly sensitive method for viral gene detection. However, the currently accepted diagnostic method based on RT-qPCR can only detect viral presence and load, and does not provide information about viral genotype. Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone. PNA probes can effectively detect a target gene by amplification and a specific melting temperature signal. It was reported that PNA probes can effectively distinguish between mismatched sequences based on their different melting temperatures in amplified PCR products. The present study reports a novel real-time RT-PCR method for simultaneous detection and genotyping of VHSV using PNA probes. The newly-developed method showed a sensitivity similar to that of the infectious titre by fish cell cultures inoculated with the virus, except for genotype IVa, where viral inoculation in cell culture showed a 10-fold higher sensitivity than the novel method. The melting point analysis to distinguish the four genotypes was performed on 80 VHSV isolates representing all known genotypes, showing that this novel real-time RT-PCR can distinguish between all VHSV genotypes without the need of further sequencing.

Immune Recognition versus Immune Evasion Systems in Zika Virus Infection

The reemergence of the Zika virus (ZIKV) infection in recent years has posed a serious threat to global health. Despite being asymptomatic or mildly symptomatic in a majority of infected individuals, ZIKV infection can result in severe manifestations including neurological complications in adults and congenital abnormalities in newborns. In a human host, ZIKV is primarily recognized by RIG-like receptors and Toll-like receptors that elicit anti-viral immunity through the secretion of type I interferon (IFN) to limit viral survival, replication, and pathogenesis. Intriguingly, ZIKV evades its host immune system through various immune evasion strategies, including suppressing the innate immune receptors and signaling pathways, mutation of viral structural and non-structural proteins, RNA modulation, or alteration of cellular pathways. Here, we present an overview of ZIKV recognition by the host immune system and the evasion strategies employed by ZIKV. Characterization of the host-viral interaction and viral disease mechanism provide a platform for the rational design of novel prophylactic and therapeutic strategies against ZIKV infection.

The Role of NS1 Protein in the Diagnosis of Flavivirus Infections.

Nonstructural protein 1 (NS1) is a glycoprotein among the flavivirus genus. It is found in both membrane-associated and soluble secreted forms, has an essential role in viral replication, and modulates the host immune response. NS1 is secreted from infected cells within hours after viral infection, and thus immunodetection of NS1 can be used for early serum diagnosis of dengue fever infections instead of real-time (RT)-PCR. This method is fast, simple, and affordable, and its availability could provide an easy point-of-care testing solution for developing countries. Early studies show that detecting NS1 in cerebrospinal fluid (CSF) samples is possible and can improve the surveillance of patients with dengue-associated neurological diseases. NS1 can be detected postmortem in tissue specimens. It can also be identified using noninvasive methods in urine, saliva, and dried blood spots, extending the availability and effective detection period. Recently, an enzyme-linked immunosorbent assay (ELISA) assay for detecting antibodies directed against Zika virus NS1 has been developed and used for diagnosing Zika infection. This NS1-based assay was significantly more specific than envelope protein-based assays, suggesting that similar assays might be more specific for other flaviviruses as well. This review summarizes the knowledge on flaviviruses' NS1's potential role in antigen and antibody diagnosis.

Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein

A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid. An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated. Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60. Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity.Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics.In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases’ active sites.

Hepatitis C Virus Nonstructural Protein 3 Increases Secretion of Interleukin-lbeta in HEK293T Cells with a Reconstructed NLRP3 Inflammasome

The pathology of diseases arising from infections by viruses of Flaviviridae is largely determined by the development of systemic inflammation. The cytokines interleukin-1beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR pyrin domain-containing family 3) inflammasomes was detected in cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or to inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, a functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural NS1 proteins of the West Nile virus, NS3 of hepatitis C virus, or NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

Structure, function and dynamics of alphavirus capping pores.

Positive-sense single stranded RNA viruses such as coronaviruses, flaviviruses or alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells. Remodelling of the host cell membranes for formation of these organelles is coupled to the membrane association of the viral replication complexes and RNA synthesis. These viral niches allow for concentration of metabolites and proteins for the synthesis of viral RNA, also preventing its detection by the cellular innate immune system. I will present the cryo-EM structural characterization of the chikungunya virus non-structural protein 1, the viral protein responsible for RNA capping and membrane binding of the viral replication machinery, and the capping mechanism. The structures show the enzyme in its active form assembled in a monotopic membrane-associated dodecameric rings. It provides the structural basis for the coupling between membrane binding and allosteric activation of the capping enzyme. We also show different stages of the reaction pathway carried out by the enzyme, showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Potential Protective Effect of Dengue NS1 Human Monoclonal Antibodies against Dengue and Zika Virus Infections.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1β, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.