Virus Nonstructural Protein Research Articles

Protein and peptide research applied to Covid-19 and SARS-Cov-2

The emergence of SARS-Cov-2 remains a challenge for the global scientific community. Many efforts have been made to rapidly develop vaccines against SARS-Cov-2. Currently available vaccines, based on the spike glycoprotein (S), are effective in generating neutralising antibodies to prevent severe acute forms of Covid-19. However, the emergence and spread of SARS-Cov-2 variants with mutations in the S protein raises questions about the need to adapt the vaccine strategy to the evolution of the virus. Proteome analysis of many SARS-Cov-2 clinical isolates suggests the development of vaccines based on peptides corresponding to epitopes located in the constant regions of viral proteins that could be effective despite the appearance of variants or the emergence of new strains of beta coronavirus. Other therapeutic innovations against Covid-19 could come from the development of peptides that inhibit structural and non-structural viral proteins. Beyond the therapeutic aspects, research on Covid-19-related proteins and peptides is also directed towards the development of diagnostic tools. The study of the glycosylation of viral proteins and the ACE-2 receptor is important for the development of therapies and diagnostics.

Molecular docking and dynamics studies of Nicotinamide Riboside as a potential multi-target nutraceutical against SARS-CoV-2 entry, replication, and transcription: A new insight

The highly contagious Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is a newborn infectious member of the dangerous beta-coronaviruses (β-CoVs) following SARS and MERS‐CoVs, can be regarded as the most significant issue afflicting the whole world shortly after December 2019. Considering CoVs as RNA viruses with a single-stranded RNA genome (+ssRNA), the critical viral enzyme RNA dependent RNA polymerase (RdRp) is a promising therapeutic target for the potentially fatal infection COVID-19. Nicotinamide riboside (NR), which is a naturally occurring analogue of Niacin (vitamin B3), is expected to have therapeutic effects on COVID-19 due to its super close structural similarity to the proven RdRp inhibitors. Thus, at the first phase of the current molecular docking and dynamics simulation studies, we targeted SARS-CoV-2 RdRp. On the next phase, SARS-CoV RdRp, human Angiotensin-converting enzyme 2, Inosine-5’-monophosphate dehydrogenase, and the SARS-CoV-2 Structural Glycoproteins Spike, Nonstructural viral protein 3-Chymotrypsin-like protease, and Papain-like protease were targeted using the docking simulation to find other possible antiviral effects of NR serendipitously. In the current study, the resulted scores from molecular docking and dynamics simulations as the primary determinative factor as well as the observed reliable binding modes have demonstrated that Nicotinamide Riboside and its active metabolite NMN can target human ACE2 and IMPDH, along with the viral Spro, Mpro, PLpro, and on top of all, RdRp as a potential competitive inhibitor.

Indole alkaloids inhibit zika and chikungunya virus infection in different cell lines

BackgroundIn recent years, an increase in the occurrence of illnesses caused by two clinically- important arboviruses has been reported: Zika virus (ZIKV) and Chikungunya virus (CHIKV). There is no licensed antiviral treatment for either of the two abovementioned viruses. Bearing in mind that the antiviral effect of indole alkaloids has been reported for other arboviral models, the present study proposed to evaluate the antiviral in vitro and in silico effects of four indole alkaloids on infections by these two viruses in different cell lines.MethodsThe antiviral effects of voacangine (VOAC), voacangine-7-hydroxyindolenine (VOAC-OH), rupicoline and 3-oxo voacangine (OXO-VOAC) were evaluated in Vero, U937 and A549 cells using different experimental strategies (Pre, Trans, Post and combined treatment). Viral infection was quantified by different methodologies, including infectious viral particles by plating, viral genome by RT-qPCR, and viral protein by cell ELISA. Moreover, molecular docking was used to evaluate the possible interactions between structural and nonstructural viral proteins and the compounds. The results obtained from the antiviral strategies for each experimental condition were compared in all cases with the untreated controls. Statistically significant differences were identified using a parametric Student’s t-test. In all cases, p values below 0.05 (p < 0.05) were considered statistically significant.ResultsIn the pre-treatment strategy in Vero cells, VOAC and VOAC-OH inhibited both viral models and OXO-VOAC inhibited only ZIKV; in U937 cells infected with CHIKV/Col, only VOAC-OH inhibited infection, but none of the compounds had activity in A549 cells; in U937 cells and A549 cells infected with ZIKV/Col, the three compounds that were effective in Vero cells also had antiviral activity. In the trans-treatment strategy, only VOAC-OH was virucidal against ZIKV/Col. In the post-treatment strategy, only rupicoline was effective in the CHIKV/Col model in Vero and A549 cells, whereas VOAC and VOAC-OH inhibited ZIKV infection in all three cell lines. In the combined strategy, VOAC, VOAC-OH and rupicoline inhibited CHIKV/Col and ZIKV/Col, but only rupicoline improved the antiviral effect of ZIKV/Col-infected cultures with respect to the individual strategies. Molecular docking showed that all the compounds had favorable binding energies with the structural proteins E2 and NSP2 (CHIKV) and E and NS5 (ZIKV).ConclusionsThe present study demonstrates that indole alkaloids are promising antiviral drugs in the process of ZIKV and CHIKV infection; however, the mechanisms of action evaluated in this study would indicate that the effect is different in each viral model and, in turn, dependent on the cell line.

Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

N130, N175 and N207 are N-linked glycosylation sites of duck Tembusu virus NS1 that are important for viral multiplication, viremia and virulence in ducklings

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication.

Although classic swine fever virus (CSFV) infection has been reported to induce autophagy, the specific induced mechanism remains unrevealed. Nonstructural protein 5A (NS5A) of CSFV is a multiphosphorylated protein with multiple functions to regulate viral replication and the host cell immune responses. Herein, we demonstrated that CSFV NS5A could induce cellular autophagy and promote viral replication. In the current study, we showed that NS5A expression significantly increased the levels of autophagy-related genes (ATGs), including light chain 3 (LC3), ATG5, and Beclin 1; conversely, degradation of P62/sequestosome 1 (SQSTM1) was observed by Western blotting. The number of autophagy-like vesicles was also obviously increased in NS5A-expressing cells, as analyzed by transmission electron microscopy (TEM). Furthermore, we observed the co-localization of the NS5A and LC3 proteins by confocal immunofluorescence analysis. Direct binding of NS5A to the autophagy-related LC3 protein was confirmed by coimmunoprecipitation in vivo and by a GST pulldown assay in vitro. Through segmentation and point mutation research on the NS5A protein, we found that the N-terminal region and the phosphorylation of amino acids 81 and 92 of the NS5A protein were essential for inducing autophagy. Finally, we demonstrated that the LC3 protein had a positive effect on CSFV replication. These findings emphasize a previously unascertained interaction relationship between NS5A and LC3 in the autophagy process. Furthermore, our research revealed a new role of CSFV NS5A, particularly its N-terminal amino acids serine 81 and serine 92, as a critical regulator of CSFV-induced autophagy and have significance for extending our understanding of the CSFV-autophagy interplay.

TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity.

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-β production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.

Linear and Continuous Flavivirus Epitopes From Naturally Infected Humans.

This manuscript is an up-to-date review of experimentally validated linear and continuous epitopes identified from arbovirus members of the Flavivirus genus. We summarized 153 immunoreactive peptides from the Dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus described in studies published from 1989 to 2020. We included peptides from structural (envelope, capsid, and pre-membrane) and nonstructural (Ns1–5) viral proteins that demonstrated relevant immunoreactivity with antibodies from naturally infected or vaccinated humans. We included peptides that demonstrated relevant reactivity features, such as indicators of disease severity related to immunological or immunopathological outcomes, differential or group diagnostic markers, immunotherapy candidates, and potential for vaccine formulation. The majority of immunoreactive peptides were described for DENV probably due to its long-lasting impact on human health and the lack of efficient vaccines and therapeutic methods. Immune landscape data regarding linear immunoreactive and continuous flavivirus peptides are still scarce, and a complete and more detailed map remains to be elucidated. Therefore, this review provides valuable data for those investigating the antibody response against flavivirus infection.

Chronic kidney disease linked to SARS-CoV-2 infection: a case report

BackgroundThe recent COVID-19 pandemic has raised concerns about patient diagnosis and follow-up of chronically ill patients. Patients suffering from chronic illnesses, concomitantly infected by SARS-CoV-2, globally tend to have a worse prognosis and poor outcomes. Renal tropism and acute kidney injury following SARS-CoV-2 infection has recently been described in the literature, with elevated mortality rates. Furthermore, patients with pre-existing chronic kidney disease, infected by SARS-CoV-2, should be monitored carefully. Here, we report the case of a 69-year-old patient with splenic marginal zone lymphoma, suffering from longstanding chronic kidney disease following SARS-CoV-2 infection.Case presentationA 69-year-old male patient previously diagnosed with pulmonary embolism and splenic marginal zone lymphoma (Splenomegaly, Matutes 2/5, CD5 negative and CD23 positive), was admitted to the hospital with shortness of breath, fever and asthenia. A nasopharyngeal swab test was performed in addition to a CT-scan, which confirmed SARS-CoV-2 infection. Blood creatinine increased following SARS-CoV-2 infection at 130 μmol/l, with usual values at 95 μmol/l. The patient was discharged at home with rest and symptomatic medical treatment (paracetamol and hydration), then readmitted to the hospital in August 2020. A kidney biopsy was therefore conducted as blood creatinine levels were abnormally elevated. Immunodetection performed in a renal biopsy specimen confirmed co-localization of SARS-CoV2 nucleocapsid and protease 3C proteins with ACE2, Lewis x and sialyl-Lewis x antigens in proximal convoluted tubules and podocytes. Co-localization of structural and non-structural viral proteins clearly demonstrated viral replication in proximal convoluted tubules in this chronically ill patient. Additionally, we observed the co-localization of sialyl-Lewis x and ACE2 receptors in the same proximal convoluted tubules. Reverse Transcriptase-Polymerase Chain Reaction test performed on the kidney biopsy was negative, with very low Ct levels (above 40). The patient was finally readmitted to the haematology department for initiation of chemotherapy, including CHOP protocol and Rituximab.ConclusionsOur case emphasizes on the importance of monitoring kidney function in immunosuppressed patients and patients suffering from cancer following SARS-CoV-2 infection, through histological screening. Further studies will be required to decipher the mechanisms underlying chronic kidney disease and the putative role of sialyl-Lewis x and HBGA during SARS-CoV-2 infection.

Cellular OCIAD2 protein is a proviral factor for hepatitis C virus replication

Hepatitis C virus (HCV) nonstructural protein NS4B is necessary for HCV replication. Our previous research found that NS4B-associated cellular proteins PREB and Surfeit 4 are involved in HCV replication. However, the molecular mechanism of HCV replication is not fully understood. Here we identified cellular ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) protein as a novel NS4B-associated HCV host cofactor by screening with small interfering RNA. Knockdown of OCIAD2 reduced significantly the HCV replication in a dose-dependent and genotype-independent manner. Further research showed that OCIAD2 was recruited into the HCV RNA replication complex by the interaction with NS4B. Interestingly, HCV replication induced OCIAD2 expression. In turn, overexpression of wild OCIAD2 also promoted virus replication whereas that of OCIAD2 mutant lacking the ability to bind NS4B exerted no effect on HCV replication. We also examined whether OCIAD2 interacted with other proteins participating in the HCV RNA replication complex including viral proteins NS5A, NS5B, and cellular proteins PREB, Surfeit 4. The results showed that OCIAD2 interacted with PREB and NS5A, but not NS5B or Surfeit 4. Our findings provide new insights into the function of OCIAD2 and HCV replication mechanism.

In silico identification of natural antiviral compounds as a potential inhibitor of chikungunya virus non-structural protein 3 macrodomain

Chikungunya Virus (CHIKV) is having a major impact on humans with potentially life-threatening and debilitating arthritis. The lack of a specific antiviral drug against the CHIKV disease has created an alarming situation to identify or develop potent chemical molecules for its remedial measures. Antiviral therapies for viral diseases are generally expensive and have adverse side effects. Plant-based antiviral natural compounds are the most suitable and best alternative of current antiviral drugs because of less toxicity. In the present study, non-structural protein 3 macrodomain (nsP3MD) of the CHIKV that is essential for virus replication has been selected for anti CHIKV drug target. The compounds were identified using molecular docking, virtual screening and further evaluated by molecular dynamics (MD) simulation studies. The binding mechanism of each compound was analyzed considering the stability and energetic parameter. We have found six plant-based natural antiviral compounds Baicalin, Rutaecarpine, Amentoflavone, Apigetrin, Luteoloside, and Baloxavir as strong inhibitors of nsP3MD of CHIKV. ADMET prediction and target analysis of the selected compounds showed drug likeliness of these compounds. MD simulation studies indicated energetically favorable complex formation between nsP3MD and the selected antiviral compounds. Furthermore, the structural effects on these substitutions were analyzed using the principles of each trajectory, which validated the interaction studies. Our analysis suggests a very high probability of these compounds to inhibit nsP3MD of CHIKV and could be evaluated for Chikungunya fever drug development. Communicated by Ramaswamy H. Sarma

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

Respiratory syncytial virus nonstructural protein 1 breaks immune tolerance in mice by downregulating Tregs through TSLP-OX40/OX40L-mTOR axis

Respiratory syncytial virus (RSV) infection in early life is associated strongly with the subsequent development and exacerbation of asthma, however, the mechanism is still ambiguous. In this study, we identified that RSV nonstructural protein (NS) 1 plays a critical role. Plasmid-mediated overexpression of NS1 induced significant airway hyperresponsiveness, eosinophilia, and mucus hyperproduction in mice. In the pNS1 group, there were markedly elevated proportions of Th2 and Th17 cells, while Th1 and Foxp3+ regulatory T cells (Tregs) significantly declined compared with the control group. Serum concentrations of interleukin (IL)-4, IL-5, IL-6, IL-17, transforming growth factor-beta, and tumor necrosis factor-alpha increased but levels of interferon-gamma and interleukin-10 declined in pNS1 group. Besides, NS1 caused a significant rise of serum thymic stromal lymphopoietin (TSLP) and OX40L levels, and a neutralizing mAb anti-OX40L was capable of promoting RSV clearance and attenuating the airway allergic inflammation caused by pNS1. Otherwise, OX40L-blocking counteracts the inhibitory effect of pNS1 on Tregs in the spleen. RSV NS1 caused elevated levels of phospho-AKT, phospho-mTOR, and phospho-S6K1, which were partially attenuated by anti-OX40L. Moreover, a specific inhibitor of mTORC1 significantly relieved the inhibition of Foxp3 expression and Tregs differentiation. Together, the data indicate that RSV NS1 protein breaks immune tolerance and induces airway inflammation and hyperresponsiveness in mice. In this process, NS1-stimulated TSLP and OX40L play a major role by inhibiting the induction of Tregs, which is at least partially mediated by modulating AKT-mTOR signaling pathways.

The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

Coxsackievirus B3 Exploits the Ubiquitin-Proteasome System to Facilitate Viral Replication.

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease.

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

Proteomic analyses identify intracellular targets for Japanese encephalitis virus nonstructural protein 1 (NS1)

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus (JEV). JEV nonstructural protein 1 (NS1) is involved in many crucial biological events during viral infection and immune suppression. To investigate the role of JEV NS1 in virus-infected cells, the molecules with which it interacts intracellularly were screened with a pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The interaction between heterogeneous nuclear ribonucleoprotein K (hnRNP K), vimentin and NS1 were verified with coimmunoprecipitation and confocal assays. Our results show that JEV NS1 interacts with vimentin, hnRNP K and colocalizes with cellular vimentin and hnRNP K. Furthermore, our results demonstrate that the expression of vimentin and hnRNP K were up-regulated in both NS1-transfected and JEV-infected cells. Knocking down vimentin and hnRNP K reduced viral replication while conversely, over-expression of vimentin and hnRNP K improved viral replication, suggesting an important role for this protein in the viral life cycle. Also, We found that vimentin also interacted with hnRNP K after overexpression of NS1 or JEV infection. These findings provide insight into the molecular mechanism of JEV replication and highlight the key role the NS1 in JEV infection.

Rational selection of hidden epitopes for a molecularly imprinted electrochemical sensor in the recognition of heat-denatured dengue NS1 protein

Rational selection of predicted peptides to be employed as templates in molecular imprinting was carried out for the heat-denatured non-structural protein 1 (NS1) of dengue virus (DENV). Conservation analysis among 301 sequences of Brazilian isolates of DENV and zika virus (ZIKV) NS1 was carried out by UniProtKB, and peptide selection was based on in silico data of the conservational, structural and immunogenic properties of the sequences. The selected peptide (from dengue 1 NS1) was synthesized and employed as a template in the electropolymerization of polyaminophenol-imprinted films on the surface of carbon screen-printed electrodes. Heat denaturation of the protein was carried out prior to analysis, in order to expose its internal hidden epitopes. After removal of the template, the molecularly imprinted cavities were able to rebind to the whole denatured protein as determined by electrochemical impedance spectroscopy. This label-free sensor was efficient to distinguish the NS1 of DENV from the NS1 of ZIKV. Additionally, the sensor was also selective for dengue NS1, in comparison with human serum immunoglobulin G and human serum albumin. Additionally, the device was able to detect the DENV NS1 at concentrations from 50 to 200 μg L−1 (RSD below 5.04%, r = 0.9678) in diluted human serum samples. The calculated LOD and LOQ were, respectively, 29.3 and 88.7 μg L−1 and each sensor could be used for six sequential cycles with the same performance.

NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

Identification of an Intramolecular Switch That Controls the Interaction of Helicase nsp10 with Membrane-Associated nsp12 of Porcine Reproductive and Respiratory Syndrome Virus.

A critical step in replication of positive-stranded RNA viruses is the assembly of replication and transcription complexes (RTC). We have recently mapped the nonstructural protein (nsp) interaction network of porcine reproductive and respiratory syndrome virus (PRRSV) and provided evidence by truncation mutagenesis that the recruitment of viral core replicase enzymes (nsp9 and nsp10) to membrane proteins (nsp2, nsp3, nsp5, and nsp12) is subject to regulation. Here, we went further to discover an intramolecular switch within the helicase nsp10 that controls its interaction with the membrane-associated protein nsp12. Deletion of nsp10 linker region amino acids 124 to 133, connecting domain 1B to 1A, led to complete relocalization and colocalization in the cells coexpressing nsp12. Moreover, single-amino-acid substitutions (e.g., nsp10 E131A and I132A) were sufficient to enable the nsp10-nsp12 interaction. Further proof came from membrane floatation assays that revealed a clear movement of nsp10 mutants, but not wild-type nsp10, toward the top of sucrose gradients in the presence of nsp12. Interestingly, the same mutations were not able to activate the nsp10-nsp2/3 interaction, suggesting a differential requirement for conformation. Reverse genetics analysis showed that PRRSV mutants carrying the single substitutions were not viable and were defective in subgenomic RNA (sgRNA) accumulation. Together, our results provide strong evidence for a regulated interaction between nsp10 and nsp12 and suggest an essential role for an orchestrated RTC assembly in sgRNA synthesis. IMPORTANCE Assembly of replication and transcription complexes (RTC) is a limiting step for viral RNA synthesis. The PRRSV RTC macromolecular complexes are comprised of mainly viral nonstructural replicase proteins (nsps), but how they come together remains elusive. We previously showed that viral helicase nsp10 interacts nsp12 in a regulated manner by truncation mutagenesis. Here, we revealed that the interaction is controlled by single residues within the domain linker region of nsp10. Moreover, the activation mutations lead to defects in viral sgRNA synthesis. Our results provide important insight into the mechanisms of PRRSV RTC assembly and regulation of viral sgRNA synthesis.