Proteins Nsp Research Articles

Differential susceptibility of human microglia HMC3 cells and brain microvascular endothelial HBEC-5i cells to Mayaro and Una virus infection

Mayaro (MAYV) and Una (UNAV) are emerging alphaviruses circulating in the Americas. Earlier reports have revealed that MAYV infects different human cell lines, including synovial and dermal fibroblasts, chondrocytes, osteoblasts, astrocytes and pericytes, as well as neural progenitor cells. In this study we evaluated the susceptibility of immortalized human microglia HMC3 cells and brain microvascular endothelial HBEC-5i cells to MAYV and UNAV infection. Cytopathic effects, cell viability, viral progeny yields, and the presence of E1 and nsP1 proteins in HMC3 and HBEC-5i cells infected with several MAYV or UNAV strains were assessed using an inverted microscope, MTT assay, plaque-forming assays, and immunofluorescence or Western blot, respectively. Finally, the expression of immune response genes was analyzed using RT-qPCR. MAYV and UNAV demonstrated strong cytopathic effects and significantly reduced cell viability in HMC3 cells. Moreover, the HMC3 cells were efficiently infected regardless of the virus strain tested, and E1 and nsP1 viral proteins were detected. In contrast, only MAYV appeared to infect HBEC-5i cells, and minimal effects on cell morphology or viability were observed. Furthermore, the MAYV titer and viral protein levels were substantially lower in the infected HBEC-5i cells when compared to those of the infected microglia cells. Finally, unlike UNAV, MAYV elicited a strong expression of specific interferon-stimulated genes in microglia cells, along with pro-inflammatory cytokines implicated in the immune response. Collectively, these findings demonstrate that MAYV and UNAV are capable of infecting relevant human brain cells.

What We Know about JN.1 (BA.2.86.1.1) SARS-CoV-2 Variant

Background: JN.1 (BA.2.86.1.1) has become the dominant strain in the U.S. at the end of 2023 according to the U.S. Centers for Disease Control and Prevention. The strain is a descendant of EG.5 family that was identified in China in February 2023 and was first detected in the United States in April 2023. JN.1* SARSCoV2 lineages: JN.1 (BA.2.86.1.1), JN.1.1, JN.1.1.1, JN.1.2, JN.1.3 and recombinants XDD (EG.5.1.1/JN.1), XDK (XBB*/JN.1.1.1). There is only a single change between JN.1 and BA.2.86 in the spike protein. JN.1 has inherited more than 30 mutations in its spike protein. It also acquired a new mutation,L455S, which further decreases the ability of antibodies to bind to the virus and prevent infection. A nonspike protein that is heavily mutated in JN.1 is the NSP3 protein. There are six mutations in NSP3 protein, namely T24I, V238L, G489S, K1155R, N1708S, and A1892T. NSP3 is one of the most active proteins in the virus, playing roles in viral RNA binding, polyprotein processing, and other functions. While the exact function of these mutations is unknown, they are likely to increase the efficiency of many of these mechanisms, creating a more functional and pathogenetic virus.The N protein is heavily mutated, R203K and G204R have been mutated in most virus variants throughout the pandemic and likely improve viral replication rate. The other mutations in N may also work to improve viral replication. While the Orf8 protein is truncated in the widespread XBB.1.5 variant, it is fully present in JN.1. The symptoms of JN.1 appear to be similar to those caused by other strains, which include sore throat, congestion, runny nose, cough, fatigue, headache, muscle aches, fever or chills, loss of sense of taste or smell, shortness of breath or difficulty breathing, nausea or vomiting and diarrhea. Some doctors have reported that upper respiratory symptoms seem to follow a pattern of starting with a sore throat, followed by congestion and a cough. Aim: The purpose of the manuscript is to present a systematic review on the prevalence, structural, genomic, and pathogenic characteristics of JN.1 from January 1 to February 29, 2024, emphasizing on the variant genetic characteristics, contagiousness, and potential pathogenicity. Material and Methods: Original scientific articles published in Medline, Pubmed, Science Direct, Web of Science, Scopus, EBSCO and BioMed Central databases, official health organizations (World Health Organization, U.S. Centers for Disease Control and Prevention, European Centre for Disease Prevention and Control) electronic publications, and specialized media in the subject, were electronically searched to accomplish the aim of the study. Articles published in any language were included from January 2024 to present using a variety of keywords in combination. The studies relevant to our review were analysed and compared. Results and Discussion: The step-change evolution of BA.2.86, combined with the immune-evading features in JN.1, has given the virus a global growth advantage well beyond the XBB.1-based lineages the world faced in 2023. Evidence suggests the human adaptive immune system could still recognise and respond to BA.286 and JN.1 effectively. Updated monovalent vaccines, tests and treatments remain effective against JN.1. There are two elements to “severity”: first if it is more ‘intrinsically’ severe (worse illness with an infection in the absence of any immunity) and second if the virus has greater transmission, causing greater illness and deaths, simply because it infects more people. The latter is certainly the case with JN.1. Conclusions: The latest data from US-CDC shows JN.1 as the prevalent SARS-CoV-2 variant in the United States. JN.1 in January 2024, quickly increase its prevalence and surpassed other variants, including HV.1 to become the most prevalent strain in The United States of América. JN.1 has a similar transmission rate, exhibits a greater evasive capacity of immune-generated antibodies than HV.1 family of SARS-CoV-2, produce similar symptoms that of other Omicron variants, are expected not to produce an increase in hospitalizations and mortality rate and the SARS-CoV-2 vaccines recently developed by Pfizer and Moderna, must be effective against this Omicron subvariant. For now, the dominant variant JN.1 does not seem harmful in terms of creating a deadly disease but is still contagious enough to not be ignored.

Emergence of crucial evidence catalyzing the origin tracing of SARS-CoV-2.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), its genetic and geographical origins remain unclear, resulting in suspicions about its natural origin. In one of our previous studies, we reported the presence of a furin cleavage site RRAR in the junction region between S1 and S2 subunits of the spike protein, which was discovered as the first crucial clue for the origin tracing of SARS-CoV-2. In the present study, we conducted an integrative analysis of new genome data from bat Sarbecovirus strains reported after the COVID-19 outbreak. The primary results included the identification of BANAL-20-52, Rp22DB159, and S18CXBatR24 as three close relatives of SARS-CoV-2 and the successful detection of seven out of nine key genomic features (designated as RC0-7 and ORF8) observed in wild types of SARS-CoV-2 in the three close relatives from Laos, Vietnam, and Yunnan province of China, respectively. The most significant contribution of the present study lies in the detection of RC1 in wild genotype in a bat Sarbecovirus population BANAL-20-52 belonging to. Encoding a segment of the NSP3 protein, RC1 was discovered as the second crucial clue for the origin tracing of SARS-CoV-2. Although RC0, encoding the junction furin cleavage site, remains undetected outside of the SARS-CoV-2 genome, Feuang of Laos is the sole place where eight of the nine wild-type features (RC1-7 and ORF8) have been detected.

Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.

Ligand Fishing Approach to Explore Amaryllidaceae Alkaloids as Potential Antiviral Candidates Targeting SARS-CoV-2 Nsp4

Ligand fishing, also described as affinity-based assay, represents a convenient and efficient approach to separate potential ligands from complex matrixes or chemical libraries. This approach contributes to the identification of lead compounds that can bind to a specific target. In the context of COVID-19, the search for novel therapeutic agents is crucial. Small molecule-based antiviral drugs, such as Amaryllidaceae alkaloids, have been described as potential candidates because they can inhibit RNA viruses. Among various SARS-CoV-2 proteins, Nsp3, Nsp4, and Nsp6 play a crucial role in the pathogenicity of the virus and are attractive targets for developing COVID-19 treatments. These proteins are responsible for the replication/transcription complex (RTC) within double-membrane vesicles (DMVs), and their inhibition disrupts the virus’s infectious cycle.Herein, we have successfully expressed and immobilized the SARS-CoV-2 Nsp4 protein on magnetic beads (Nsp4-MBs) and employed a ligand fishing assay to screen a collection of ten Amaryllidaceae-based alkaloids and applied to Hippeastrum aulicum extract. Remarkably, four out of ten alkaloids, namely 2-α-7-dimethoxyhomolycorine (6), hemanthamine (5), albomaculine (8), and tazettine (9), exhibited selective affinities for Nsp4. Albomaculine (8) and hemanthamine (5) were also identified from extract by the affinity assay. These findings highlight the potential of these alkaloids as model compounds for future drug discovery studies aimed at developing therapeutic interventions against SARS-CoV-2 infections.

Suppression of TRIM19 by arterivirus nonstructural protein 1 promotes viral replication

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

In silico evaluation of S-adenosyl-L-homocysteine analogs as inhibitors of nsp14-viral cap N7 methyltranferase and PLpro of SARS-CoV-2: synthesis, molecular docking, physicochemical data, ADMET and molecular dynamics simulations studies

A series of S-adenosyl-L-homosysteine (SAH) analogs, with modification in the base and sugar moiety, have been designed, synthesized and screened as nsp14 and PLpro inhibitors of severe acute respiratory syndrome corona virus (SARS-CoV-2). The outcomes of ADMET (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) studies demonstrated that the physicochemical properties of all analogs were permissible for development of these SAH analogs as antiviral agents. All molecules were screened against different SARS-CoV-2 targets using molecular docking. The docking results revealed that the SAH analogs interacted well in the active site of nsp14 protein having H-bond interactions with the amino acid residues Arg289, Val290, Asn388, Arg400, Phe401 and π-alkyl interactions with Arg289, Val290 and Phe426 of Nsp14-MTase site. These analogs also formed stable H-bonds with Leu163, Asp165, Arg167, Ser246, Gln270, Tyr274 and Asp303 residues of PLpro proteins and found to be quite stable complexes therefore behaved as probable nsp14 and PLpro inhibitors. Interestingly, analog 3 showed significant in silico activity against the nsp14 N7 methyltransferase of SARS-CoV-2. The molecular dynamics (MD) and post-MD results of analog 3 unambiguously established the higher stability of the nsp14 (N7 MTase):3 complex and also indicated its behavior as probable nsp14 inhibitor like the reference sinefungin. The docking and MD simulations studies also suggested that sinefungin did act as SARS-CoV-2 PLpro inhibitor as well. This study’s findings not only underscore the efficacy of the designed SAH analogs as potent inhibitors against crucial SARS-CoV-2 proteins but also pinpoint analog 3 as a particularly promising candidate. All the study provides valuable insights, paving the way for potential advancements in antiviral drug development against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

GETV nsP2 plays a critical role in the interferon antagonism and viral pathogenesis

Getah virus (GETV) was becoming more serious and posing a potential threat to animal safety and public health. Currently, there is limited comprehension regarding the pathogenesis and immune evasion mechanisms employed by GETV. Our study reveals that GETV infection exhibits the capacity for interferon antagonism. Specifically, the nonstructural protein nsP2 of GETV plays a crucial role in evading the host immune response. GETV nsP2 effectively inhibits the induction of IFN-β by blocking the phosphorylation and nuclear translocation of IRF3. Additionally, GETV nsP2 hinders the phosphorylation of STAT1 and its nuclear accumulation, leading to significantly impaired JAK-STAT signaling. Furthermore, the amino acids K648 and R649, situated in the C-terminal region of GETV nsP2, play a crucial role in facilitating nuclear localization. Not only do they affect the interference of nsP2 with the innate immune response, but they also exert an influence on the pathogenicity of GETV in mice. In summary, our study reveals novel mechanisms by which GETV evades the immune system, thereby offering a foundation for comprehending the pathogenic nature of GETV.7nE7h6WMi98wNyyQdFNuFQVideo

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Inhibition of SARS–CoV–2 NSP–15 by Uridine–5′–Monophosphate Analogues Using QSAR Modelling, Molecular Dynamics Simulations, and Free Energy Landscape

SARS–CoV–2 is accountable for severe social and economic disruption around the world causing COVID–19. Non–structural protein–15 (NSP15) possesses a domain that is vital to the viral life cycle and is known as uridylate–specific endoribonuclease (EndoU). This domain binds to the uridine 5′–monophosphate (U5P) so that the protein may carry out its nativeactivity. It is considered a vital drug target to inhibit the growth of the virus. Thus, in this current study, ML–based QSAR and virtual screening of U5P analogues targeting Nsp15 were performed to identify potential molecules against SARS–CoV–2. Screening of 816 unique U5P analogues using ML–based QSAR identified 397 compounds ranked on their predicted bioactivity (pIC50). Further, molecular docking and hydrogen bond interaction analysis resulted in the selection of the top three compounds (53309102, 57398422, and 76314921). Molecular dynamics simulation of the most promising compounds showed that two molecules 53309102 and 57398422 acted as potential binders of Nsp15. The compound was able to inhibit nsp15 activity as it wassuccessfully bound to the active site of the nsp15 protein. This was achieved by the formation of relevant contacts with enzymatically critical amino acid residues (His235, His250, and Lys290). Principal component analysis and free energy landscape studies showed stable complex formation while MM/GBSA calculation showed lower binding energies for 53309102 (ΔGTOTAL = –29.4 kcal/mol) and 57398422 (ΔGTOTAL = –39.4 kcal/mol) compared to the control U5P (ΔGTOTAL = –18.8 kcal/mol). This study aimed to identify analogues of U5P inhibiting the NSP15 function that potentially could be used for treating COVID–19.

Computational exploration of Andrographis paniculata herb compounds as potential antiviral agents targeting NSP3 (6W02) and NSP5 (7AR6) of SARS-COV-2

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, requires an urgent search for effective antiviral agents. NSP3 and NSP5, play critical roles in the replication and transcription. This study aimed to screening the physicochemical properties, toxicity, and antiviral potential of herbal compounds from Andrographis paniculata against NSP3 (6W02) and NSP5 (7AR6) of SARS-CoV-2. The herb compounds were obtained from the KNApSAcK Family and PubChem databases. Their interactions with Remdesivir, an FDA-approved antiviral for SARS-CoV-2, NSP3 and NSP5 were analyzed using molecular docking through Molegro Virtual Docker 6.0. There are 27 compounds out of a total of 41 herb compounds that meet the Veber Rule and have predicted good physicochemical properties. Moreover, 15 herb compounds predicted to be non-toxic based on GHS, not-mutagenic, not-carcinogenic, and not-allergenic to the skin. The screening results using PASS Online showed 5 compounds highly potential to be candidates for SARS-CoV-2 antivirals, namely: 12S-Hydroxyandrographolide, 14-Deoxy-11,12-didehydroandrographolide, Andrographolide, Andropanolide, and Isoandrographolide. Based on molecular docking results, it was found both Remdesivir and the herb compounds still required more energy than the native ligand on the 6W02 target protein. In contrast, for the 7AR6 target protein, both Remdesivir and herb compounds required less energy than the native ligand. In conclusion, A. paniculata herb compounds showed inhibitory activity on 6W02 and 7AR6 receptors, suggesting their potential as herbal treatments for SARS-CoV-2 antivirals by targeting the NSP3 and NSP5 proteins. Further validation through in vitro and in vivo studies is needed.

A Molecular Docking and Dynamic Simulation Study for the Inhibition of Porcine Coronavirus: Drug Repurposing Approach

Severe acute respiratory syndrome (SARS) coronavirus-2 was declared as world pandemic by WHO in 2019. It has been spreading very rapidly around the world and is responsible for various health issues. Different forms of corona viruses are capable of initiating infections in humans ranging from common cold to respiratory disorders including diarrhea. In this study, we have utilized the drug repurposing approach for identifying the therapeutic potential candidate for the inhibition of the nsp1 protein of a new porcine coronavirus 6lpa. 81 FDA approved antiviral agents were docked against this virus using discovery studio and PyRx software. Analysis of result reveals three compounds based on the best docking score: Abivertinib, Vazegepant and Elbasvir. Pharmacological and toxicological properties of the leads were also determined. Furthermore, molecular dynamics simulation studies were performed to determine the stability of complexes. RMSD of protein- ligand complex is showing excellent stability with all three drugs. Least conformational changes can be seen within Abivertinib complex which shows its stability during simulation process. These screened drugs obtained through drug repurposing approach can prove to be useful in treating the infections caused by porcine corona virus 6lpa after further studies.

The repurposing of FDA-approved drugs to stop viral replication in COVID-19 treatment: a comprehensive molecular docking and dynamics analysis

Amidst a public health crisis such as the SARS-CoV-2 induced COVID-19 pandemic, the urgency to develop and provide access to novel drugs becomes paramount. In these scenarios, repurposing existing drugs emerges as an appealing strategy due to their established safety profiles and prior regulatory approvals, potentially streamlining their adoption for new therapeutic purposes. There has been a notable interest in investigating the repurposing potential of FDA-approved drugs to combat the disease triggered by the SARS-CoV-2 virus. This investigation was conducted with the objective of identifying FDA-approved antiviral drugs that could target both the original and mutant forms of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) protein, given the escalating prevalence of SARS-CoV-2 mutations that undermined vaccine efficacy and underscored the need for alternative treatment avenues. Despite ongoing clinical trials, the selection of drugs specifically designed to combat SARS-CoV-2 remains limited. The present study aimed to assess the inhibitory potential of eight approved antiviral drugs. The wild-type and mutant (P323L) RdRp protein (PDB ID: 7BV2). By utilizing molecular docking techniques, the selected drugs were evaluated based on their binding affinities with both the mutant and wild-type RdRp protein. Subsequently, the protein-ligand complexes exhibiting the highest binding affinities were subjected to a 150 ns molecular dynamics simulation to evaluate their stability. The outcomes of the molecular docking analysis pinpointed Dolutegravir and Beclabuvir as possessing the most robust binding affinities for both mutant and wild-type RdRp systems. These findings prompted the selection of these drugs for an in-depth molecular dynamic simulation (MDS) investigation. The comprehensive analysis of Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (Rg), Solvent Accessible Surface Area (SASA) and Secondary Structure Dynamics confirmed that Dolutegravir and Beclabuvir are potent inhibitors of the NSP12 protein. Collectively, the computational assessments put forth two potential contenders: Dolutegravir and Beclabuvir as promising inhibitors against both the wild-type and mutant RdRp proteins. To further validate these drug candidates, rigorous in vitro and in vivo investigations are warranted which could eventually position them as valuable therapeutic agents in the fight against COVID-19.

Rotavirus capping enzyme VP3 inhibits interferon expression by inducing MAVS degradation during viral replication.

Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

Exploration of 1,2,3-triazolo fused triterpenoids as inhibitors of human coronavirus 229E targeting the viral nsp15 protein.

The coronavirus disease-19 (COVID-19) pandemic has raised major interest in innovative drug concepts to suppress human coronavirus (HCoV) infections. We previously reported on a class of 1,2,3-triazolo fused betulonic acid derivatives causing strong inhibition of HCoV-229E replication via the viral nsp15 protein, which is proposedly related to compound binding at an intermonomer interface in hexameric nsp15. In the present study, we further explored the structure-activity relationship (SAR), by varying the substituent at the 1,2,3-triazolo ring as well as the triterpenoid skeleton. The 1,2,3-triazolo fused triterpenoids were synthesized by a multicomponent triazolization reaction, which has been developed in-house. Several analogs possessing a betulin, oleanolic acid, or ursolic acid core displayed favorable activity and selectivity (EC50 values for HCoV-229E: 1.6-3.5 μM), but neither of them proved as effective as the lead compound containing betulonic acid. The 18β-glycyrrhetinic acid-containing analogs had low selectivity. The antiviral findings were rationalized by in silico docking in the available structure of the HCoV-229E nsp15 protein. The new SAR insights will aid the further development of these 1,2,3-triazolo fused triterpenoid compounds as a unique type of coronavirus inhibitors.

The replicase protein nsp2 of Chinese highly pathogenic porcine reproductive and respiratory virus is involved in protective immunity by promoting viral clearance

The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus (PRRSV), and our previous studies have shown that the PRRSV nsp2 genetic variation contributes to poor cross-neutralization. By using in vitro antibody absorption assay, here we show that the papain-like protease 2 (PLP2) domain of nsp2 is a target of neutralizing antibodies. This was further verified by cross-neutralization assay with a series of inter-lineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 and the low virulent NADC30-like strain CHsx1401 (lineage 1). The role of nsp2 in protective immunity was subsequently tested in a one-month SPF piglet model by immunizing the piglets with CHsx1401 or its derivatives carrying JXwn06 structural protein region (SP) alone (CHsx1401-SPJX) or in combination with PLP2 region (CHsx1401-SPplp2JX), or the whole nsp2 region (CHsx1401-SPnsp2JX), followed by challenge with JXwn06 at 42 days post immunization, a time point when the viremia was undetectable. All chimera groups were protected from the challenge by JXwn06, whereas the group CHsx1401 failed to provide beneficial protection. Interestingly, the group CHsx1401-SPnsp2JX, but not CHsx1401-SPplp2JX, showed the lowest lung microscopic lesions and viral tissue load. Significantly, the vaccine virus CHsx1401-SPnsp2JX was undetectable in the examined tissues, and so was for the challenge virus except for one piglet, highlighting an important role of HP-PRRSV nsp2 in promoting viral clearance. The findings provide insight into the mechanisms underlying the protective immunity against PRRSV and have important implications in PRRSV vaccine development.

First person – Parisa Naeli

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Parisa Naeli is first author on ‘ The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression’, published in JCS. Parisa is a postdoctoral researcher in the lab of Seyed Mehdi Jafarnejad at Queen's University Belfast, UK, investigating mRNA translation and decay, RNA biology, and RNA-binding proteins.

Potential Alphavirus Inhibitors From Phytocompounds – Molecular Docking and Dynamics Based Approach

Background. Alphaviral diseases are an economic burden all over the world due to their chronicity and distribution worldwide. The glycoproteins E1 and E2 are important for binding to the surface of the host cell by interacting with the receptors and non-structural proteins named nsP2 and nsP4 are important for the replication of virus, so can be an important drug discovery target. Objective. We are aimed to explore the in silico interaction between plant-based compounds (phytocompounds) and specific protein targets, such as nonstructural protein nsP4 and glycoprotein E2 of Sindbis virus (SINV), nsP2 and E2 of Chikungunya virus (CHIKV), and glycoproteins E1 and E2 of Ross River virus (RRV). Methods. A library of phytochemicals from Indian medicinal plants was prepared using databases and converted to 3D structures. Protein structures (nsP2, nsp4, E1, E2) were obtained and refined, followed by molecular docking with AutoDock Vina. Promising ligands were evaluated for properties, cytotoxicity, and mutagenicity, considering drug-likeness and potential issues. Molecular Dynamics simulations assessed complex stability. Results. We analyzed 375 phytocompounds against these targets using molecular docking, modeling, and molecular dynamics for SINV, CHIKV, and Ross River (RRV) virus proteins. Granatin A has been found to successfully bind to the target sites of SINV nsP4, CHIKV E2, and CHIKV nsP2 with binding affinity values of -16.2, -20.6, and -18.6 Kcal/mol respectively. Further, stability of CHIKV E2 – Granatin A complex was done by performing molecular dynamic simulation and the complex was stable at 60ps. Conclusions. This research provides valuable insights into the development of effective antiviral drugs against alphaviruses, emphasizing the importance of natural compounds and their interactions with viral proteins. This study might pave the way for further exploration of these small molecules as effective anti-alphaviral therapeutic agents.

Antiviral evaluation of 1,4-disubstituted-1,2,3-triazole derivatives against Chikungunya virus.

This work aimed to investigate the antiviral activity of two 1,4-disubstituted-1,2,3-triazole derivatives (1 and 2) against Chikungunya virus (CHIKV) replication. Cytotoxicity was analyzed using colorimetric assays and the antiviral potential was evaluated using plaque assays and computational tools. Compound 2 showed antiviral activity against CHIKV 181-25 in BHK-21 and Vero cells. Also, this compound presented a higher activity against CHIKV BRA/RJ/18 in Vero cells, like compound 1. Compound 2 exhibited virucidal activity and inhibited virus entry while compound 1 inhibited virus release. Molecular docking suggested that these derivatives inhibit nsP1 protein while compound 1 may also target capsid protein. Both compounds exhibit promising antiviral activity against CHIKV by blocking different steps of virus replication.