Virus Nonstructural Protein Research Articles

The Zika virus NS5 protein binds HSP90 to suppress EGF-induced Akt signaling and trophoblast cell migration.

Zika virus (ZIKV) infection during pregnancy can cause congenital Zika virus syndrome (CZV), including fetal growth restriction and death. In the developing placenta, trophoblast cells respond to epidermal growth factor (EGF) to migrate into the decidua to facilitate implantation and fetal development. EGF activates the Akt protein kinase, a master regulator of trophoblast cell migration. Akt signaling and stability are dependent on heat shock protein 90 (HSP90), which mediates the maturation of proteins necessary for EGF/Akt signaling. Here we show that ZIKV infection inhibits EGF-mediated Akt activation and downstream signaling to suppress trophoblast migration. The ZIKV non-structural protein 5 (NS5) is sufficient to inhibit trophoblast migration through its binding interaction with HSP90, leading to suppression of Akt phosphorylation and inhibition of EGF-induced trophoblast migration. Thus, ZIKV NS5/HSP90 interactions play a key role in disruption of trophoblast function, revealing an underlying cause of improper placental development and fetal disease.

Domperidone inhibits dengue virus infection by targeting the viral envelope protein and nonstructural protein 1

Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection, which remains a major public health concern worldwide owing to the lack of specific treatments or antiviral drugs available. This study investigated the potential repurposing of domperidone, an antiemetic and gastrokinetic agent, to control DENV infection. Domperidone was identified by pharmacophore-based virtual screening as a small molecule that can bind to both the viral envelope (E) and the nonstructural protein 1 (NS1) of DENV. Molecular dynamics (MD) simulations and surface plasmon resonance (SPR) analysis were subsequently performed to determine specific interactions of domperidone with the DENV E and NS1 proteins and their binding affinity. Treatment of immortalized human hepatocyte-like cells (imHC) with domperidone could inhibit DENV production and NS1 secretion in a dose-dependent manner following infection with DENV serotype 2. These inhibitory effects were mediated by reduction in viral RNA replication and viral E and NS1 protein expression, but not by interference with virus entry into cells or NS1 oligomerization. The suppression of DENV production and NS1 secretion by domperidone was observed across all four DENV serotypes to varying degrees between different virus strains. The findings from our study suggest viral target-based repurposing of domperidone for modulating DENV.

The Chikungunya Virus nsP3 Macro Domain Inhibits Activation of the NF-κB Pathway

The role of the chikungunya virus (CHIKV) non-structural protein 3 (nsP3) in the virus lifecycle is poorly understood. The protein comprises three domains. At the N-terminus is a macro domain, biochemically characterised to bind both RNA and ADP-ribose and to possess ADP-ribosyl hydrolase activity—an enzymatic activity that removes ADP-ribose from mono-ADP-ribosylated proteins. As ADP-ribosylation is important in the signalling pathway, leading to activation of the transcription factor NF-κB, we sought to determine whether the macro domain might perturb NF-κB signalling. We first showed that CHIKV infection did not induce NF-κB activation and could not block exogenous activation of the pathway via TNFα, although TNFα treatment did result in a modest reduction in virus titre. In contrast, ectopic expression of nsP3 was able to inhibit both basal and TNFα-mediated NF-κB activation, and this was dependent on the macro domain, as a mutation previously shown to disrupt ADP-ribose binding and hydrolase activity (D10A) eliminated the ability to inhibit NF-κB activation. The macro domain D10A mutant also resulted in a dramatic reduction in virus infectivity, consistent with the notion that the ability of the macro domain to inhibit NF-κB activation plays a role in the virus lifecycle.

Conserved Nuclear Localization Signal in NS2 Protein of Bombyx Mori Bidensovirus: A Potential Invertebrate ssDNA Virus Trait.

Bombyx mori bidensovirus (BmBDV), a significant pathogen in the sericulture industry, holds a unique taxonomic position due to its distinct segmented single-stranded DNA (ssDNA) genome and the presence of a self-encoding DNA polymerase. However, the functions of viral non-structural proteins, such as NS2, remain unknown. This protein is hypothesized to play a role in viral replication and pathogenesis. To investigate its structure and function, we employed phylogenetic analysis, subcellular localization, mutational analysis, and a dual-luciferase reporter system to characterize the nuclear localization signal (NLS) within NS2 and its effect on viral promoter activity. Additionally, co-immunoprecipitation and mass spectrometry were utilized to identify host proteins interacting with NS2. We identified a functional bipartite NLS in NS2, validated the combination pattern of key amino acids, and demonstrated its role in regulating viral promoter activity. Furthermore, we identified potential NLSs in NS2 homologs in other invertebrate ssDNA viruses based on sequence analysis. We also revealed interactions between NS2 and host nuclear transport proteins, suggesting that it plays a role in nuclear transport and viral replication. This research underscores the importance of NS2's NLS in BmBDV's life cycle and its potential conservation across invertebrate ssDNA viruses, providing insights into virus-host interactions and avenues for antiviral strategy development.

Humoral and cellular response to SARS-CoV-2 mRNA vaccine in paediatric heart transplant recipients.

The aim of this prospective cohort study is to analyse the humoral and cellular vaccine responses in paediatric heart transplant recipients (HTR, n=12), and compare it with the response in healthy controls (HC, n=14). All participants were 5-18 years old and vaccinated with mRNA vaccine against SARS-CoV-2 between December 2021 and May 2022. The humoral response was measured by quantifying antibody titers against SARS-CoV-2 spike protein (anti-S). The T-lymphocyte phenotype and SARS-CoV2-specific CD4+ and CD8+ T-cell response was studied by multiparametric flow cytometry through peripheral blood mononuclear cells by the quantification of degranulation markers (CD107a) and intracellular cytokines (IFN-γ, TNF-α and IL-2) after in vitro stimulation with SARS-CoV-2 peptides from structural proteins (S, M, N, E) and non-structural viral proteins. After vaccination, humoral response was found in all HTR, although they showed lower levels of anti-S IgG compared to HC (p=0.003). However, in terms of cellular response, no significant differences were obtained in the prevalence of responders and magnitude of responses between groups. In addition, anti-S IgG levels directly correlated with a higher SARS-CoV-2 specific T-cell response (rho=0.43; p=0.027 and rho=0.45; p=0.02 for IFN-γ+ and TNF-α+ production of CD8+ T-cells, respectively). Activated T-cell phenotype in HTR was associated with a lower humoral response to SARS-CoV-2 vaccine. HTR had humoral response after vaccination, although they showed lower levels of specific anti-S antibodies compared to HC. There were no significant differences in the SARS-CoV2-specific cellular response between the two groups. Obtaining satisfactory data on this type of response could potentially challenge the current vaccine guideline recommendations.

Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell.

Translation errors, impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal, including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways, the ubiquitin-proteasome system and selective autophagy. In humans, the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain-Barré syndrome). Non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses, catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm, ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here, we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell, although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells, while transient overexpression of UBA1 favours faster replication kinetics, with higher virus titres and protein levels detected. Moreover, UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.

RTP4 restricts influenza A virus infection by targeting the viral NS1 protein.

The influenza A virus evades the host innate immune response to establish infection by inhibiting RIG-I activation through its nonstructural protein 1 (NS1). Here, we reported that receptor-transporting protein 4 (RTP4), an interferon-stimulated gene (ISG), targets NS1 to inhibit influenza A virus infection. Depletion of RTP4 significantly increased influenza A virus multiplication, while NS1-deficient viruses were unaffected. Mechanistically, RTP4 interacts with NS1 in an RNA-dependent manner and sequesters it from the TRIM25-RIG-I complex, thereby restoring TRIM25-mediated RIG-I K63-linked ubiquitination and subsequent activation of IRF3. Antiviral activity of RTP4 requires the evolutionarily conserved CXXC motifs and an H149 residue in the zinc finger domain, mutations of which disrupted RTP4-NS1 interaction and abrogated the ability of RTP4 to rescue RIG-I-mediated signaling. Collectively, our findings provided insights into the mechanism by which an ISG restricts influenza A virus replication by reactivating host antiviral signaling.

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Tertiary Structures of Haseki Tick Virus Nonstructural Proteins Are Similar to Those of Orthoflaviviruses.

Currently, a large number of novel tick-borne viruses potentially pathogenic to humans are discovered. Studying many of them by classical methods of virology is difficult due to the absence of live viral particles or a sufficient amount of their genetic material. In this case, the use of modern methods of bioinformatics and synthetic and structural biology can help. Haseki tick virus (HSTV) is a recently discovered tick-borne unclassified ssRNA(+) virus. HSTV-positive patients experienced fever and an elevated temperature. However, at the moment, there is no information on the tertiary structure and functions of its proteins. In this work, we used AlphaFold 3 and other bioinformatic tools for the annotation of HSTV nonstructural proteins, based on the principle that the tertiary structure of a protein is inextricably linked with its molecular function. We were the first to obtain models of tertiary structures and describe the putative functions of HSTV nonstructural proteins (NS3 helicase, NS3 protease, NS5 RNA-dependent RNA-polymerase, and NS5 methyltransferase), which play a key role in viral genome replication. Our results may help in further taxonomic identification of HSTV and the development of direct-acting antiviral drugs, POC tests, and vaccines.

Flaviviruses induce ER-specific remodelling of protein synthesis.

Flaviviruses orchestrate a unique remodelling of the endoplasmic reticulum (ER) to facilitate translation and processing of their polyprotein, giving rise to virus replication compartments. While the signal recognition particle (SRP)-dependent pathway is the canonical route for ER-targeting of nascent cellular membrane proteins, it is unknown whether flaviviruses rely on this mechanism. Here we show that Zika virus bypasses the SRP receptor via extensive interactions between the viral non-structural proteins and the host translational machinery. Remarkably, Zika virus appears to maintain ER-localised translation via NS3-SRP54 interaction instead, unlike other viruses such as influenza. Viral proteins engage SRP54 and the translocon, selectively enriching for factors supporting membrane expansion and lipid metabolism while excluding RNA binding and antiviral stress granule proteins. Our findings reveal a sophisticated viral strategy to rewire host protein synthesis pathways and create a replication-favourable subcellular niche, providing insights into viral adaptation.

Dengue virus non-structural protein 1 binding to thrombin as a dengue severity marker: Comprehensive patient analysis in south Taiwan.

Previously we identified a complex of non-structural protein (NS) 1 - Thrombin (NST) in dengue infected patients. Here, we investigated how the concentration of NS1 and NST differ in various dengue severity levels as well as their demographic and clinical features. Several comorbid (hypertension, diabetes, and chronic renal failure) often found in dengue patients were also measured and analyzed. A total of 86 dengue patients (52 not severe and 34 severe), were enrolled and had their blood taken. Blood samples were further verified for clinical blood parameters, including liver and renal function tests and serologic assays (NS1 and NST). Patients' severity was grouped based on WHO 2009 classification, which separates patients into dengue without warning signs (DNWS), dengue with warning signs (DWWS), and severe dengue (SD). DWWS is explained as DNWS with warning signs (persistent abdominal pain, persistent vomiting, liver enlargement, bleeding (any kind), fatigue, and restlessness). SD are those with severe plasma leakage, severe bleeding, or severe organ impairment. Multivariate regression analysis was used to predict the role of NST on the dengue severity development and receiver operating characteristic (AUROC) test was utilized to evaluate separability. The analysis revealed that NS1 significantly impacts the disease outcome (p 0.018, OR=2.467 (1.171-5.197)) but not beyond the effect through NST (p 0.108, OR=0.085 (0.004-1.719)). We also prove that NST was a better severity biomarker compared to NS1, as it can predict progression from DNWS to DWWS (AUC: NS1=0.771∗∗, NST=0.81∗∗) and SD (AUC: NS1=0.607, NST=0.754∗) significantly. This finding suggests the importance of NST in mediating the NS1 effect to promote dengue severity progression and its promising capability as an acute stage dengue severity biomarker.

A multiscale in silico approach to impede the interaction between Dengue NS1 and Human RPL18, aiming to suppress the Dengue viral translation and proliferation

ABSTRACT The Non-Structural protein 1 (NS1) of the Dengue virus is a crucial viral protein that plays a key role in host evasion and viral replication. The present study explores the pathophysiology of the host during the interaction with the viral protein via computational methods like protein–protein docking, virtual screening, and molecular dynamic simulation. Therefore, in this study we have focused on Dengue NS1 and the Human RPL18, to explore the binding mechanism and study their importance in viral proliferation. The results provided an insight into the vital amino acids of DENV NS1 (Gln_70 Glu_74 and Gly_161) which are responsible for viral-host interaction. The screening results showed that the Enamine_2436, NCI_99799, Malarial_19966, and FDA_54, had the strongest interaction with a docking score of −10.71, −8.11, −7.78, and −7.19 kcal/mol. In addition to the screening, the efficacy of the leads was validated through HOMO–LUMO studies. The Molecular dynamics simulation, PCA/FEL, and MMPBSA analysis showed that the Malarial_19966 maintained overall stability and binding affinity when compared to other leads. Hence the study comprehensively provides an insight into the biological phenomena of Dengue virus NS1 binding with Human RPL18 protein, along with possible inhibitors to suppress the Dengue viral translation and proliferation.

Enhancement of systemic and lung-localized CD4<sup>+</sup> T-cell immune responses by truncation of NS1 protein of a seasonal live influenza vaccine strain

Introduction. There is a large variety of licensed influenza vaccines worldwide, but their common limitation is rather narrow specificity and inability to protect against antigenic-drift variants of influenza virus. Therefore, optimization of immunogenic and cross-protective properties of licensed influenza vaccines is an urgent priority of public health agenda. One such approach is to modulate the immunogenic properties of live attenuated influenza vaccine (LAIV) by truncating the open reading frame of influenza virus non-structural protein 1 (NS1). The main objective of this study is to evaluate the immunogenic properties of the H1N1 seasonal LAIV strain by truncation of the NS1 protein to 126 amino acides. Materials and methods. Using reverse genetics technique, two H1N1 LAIV strains with full-length and truncated NS1 protein with three consecutive stop codons added after the 126th amino acid residue were obtained.C57BL/6J mice were immunized intranasally with the vaccine candidates, twice at a three-week interval. Seven days after the second immunization, cells were isolated from spleen and lung tissues and stimulated with whole wild-type H1N1 influenza virus. Levels of systemic and tissue-resident cytokine-producing CD4+ and CD8+ memory T cells were assessed by intracellular cytokine staining assay with flow cytometry. Replication of engineered vaccine strains in in vitro and in vivo systems was also evaluated. Results. Truncation of NS1 protein of the LAIV strain significantly increased the levels of virus-specific CD4+ effector memory T cells in spleens and the levels of CD4+ tissue-resident memory T cells in lungs of mice after two-dose immunization, indicating a higher potential for protection against influenza infection of the LAIV NS126 vaccine strain compared to the classical variant of LAIV. Importantly, the LAIV NS126 strain also had a more pronounced attenuated phenotype in mice than its classical counterpart.

Investigating the effect of linker peptides on the fragmentation of Fc-fusion proteins in transient gene expression of mammalian cells

Protein fragmentation is a critical quality attribute for Fc-fusion protein production in mammalian cells. In the production of viral non-structural proteins as the form of Fc-fusion protein, fragmentation of Fc-fusion proteins occurred in two transient gene expression (TGE) systems with human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. The introduction of a flexible empirical linker reduced fragmentation in HEK293 cells, but not in CHO cells. Additionally, two rigid empirical linkers failed to restore impaired Fc-fusion proteins in CHO cells. In vitro incubation assay using conditioned culture medium and cultures supplemented with protease inhibitor cocktail suggest that fragmentation of Fc-fusion proteins in CHO cells may be due to various host cell proteins released into the culture medium. These findings suggest that the introduction of linker peptides can improve the fragmentation of Fc-fusion proteins in mammalian cells, but exhibit different fragment patterns depending on the cell type.

A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles.

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, resulting in broad-spectrum anticoronaviral activity.

Chromatin mimicry by human JC virus.

Chronically persistent viruses are integral components of the organismal ecosystem in humans and animals 1 2 . Many of these viruses replicate and accumulate within the cell nucleus 3 . The nuclear location allows viruses to evade cytoplasmic host viral sensors and promotes viral replication 4 . One of the unexplored and puzzling aspects of the viral nuclear lifecycle involves the virus's ability to deal with the physical constraints of nuclear architecture. To replicate and accumulate within the nucleus in large numbers sufficient for infection spreading, DNA viruses need to overcome the spatial limitations imposed by chromatin and the nuclear matrix. We found that one of the most widespread and potentially lethal human viruses, the JC polyomavirus 5 , interferes with nuclear heterochromatin to create virus-occupied space. The JC virus's impact on heterochromatin is mediated by the viral nonstructural protein, Agnoprotein (Agno). Agno's interference with heterochromatin is governed by structurally diverse mimics of host epigenetic regulators that facilitate virus-induced chromatin reorganization and a dramatic decline in nuclear stiffness in the infected cells. The JCV epigenetic mimicry is critical for the virus infection, as evident from reduced replication of mimic-mutant viruses. Our data suggest that modulation of nuclear mechanical properties is a novel strategy enabling chronicity of the JC and possibly other nuclear virus infections.

Unraveling the impact of mutations on the functional dynamics, stability, and energetics of dengue virus non-structural protein 1

ABSTRACT The non-structural protein 1 (NS1), a viral toxin, is linked to serious consequences of dengue. Given its crucial role in the viral life cycle, NS1 has been a primary target for antiviral drug development. This study elucidates the mechanistic impact of specific NS1 mutations, V220D and A248V, identified by Tan and colleagues as potential inhibitors of viral replication. We developed an integrated computational framework to analyze these mutations relative to the native NS1 protein. Using conventional all-atom molecular dynamic simulations followed by site-directed in silico mutagenesis, significant alterations in protein structure were observed. Conformational ensembles over time showed average backbone deviations of 0.44 nm and 0.43 nm for V220D and A248V, respectively, compared to 0.37 nm for the wild type. Residue-wise fluctuations were higher in A248V compared to both wild type and V220D systems. Mutation-induced disruptions in the hydrogen bond network and altered protein structure graphs were also revealed. Principal component analysis and free energy landscape assessments showed the wild-type protein had a more constrained and stable conformation, whereas the mutants displayed scattered energy contours, suggesting reduced structural stability. This comprehensive assessment provides deep insight into the structural impact of NS1 mutations, informing future efforts in antiviral drug development targeting NS1.

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 promotes the autophagic degradation of adaptor protein SH3KBP1 to antagonize host innate immune responses by enhancing K63-linked polyubiquitination of RIG-I.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third polyproline-arginine motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for developing novel attenuated vaccines against PRRSV.

Non-lytic spread of poliovirus requires the nonstructural protein 3CD.

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread.

Broad-Spectrum Antiviral Agents against SARS-CoV-2 Variants Inhibit the Conserved Viral Protein Nsp1-RNA Interaction.

The ongoing global threats posed by COVID-19 pandemic, catalyzed by SARS-CoV-2, underscores the pressing need for effective antiviral strategies. The viral non-structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem-loop 1 (SL1) in the 5' untranslated region (UTR). We have developed a novel dual-luciferase reporter assay, designed to investigate the critical Nsp1-SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1-40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID-19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.