Nonstructural Protein Research Articles

Nanobody against SARS-CoV-2 non-structural protein Nsp9 inhibits viral replication in human airway epithelia

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-CoV-2 entry in the host cell by targeting surface-exposed Spike protein. Here we have established a pipeline that instead targets highly conserved viral proteins made only after viral entry into the host cell when the SARS-CoV-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. One of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was expressed and found to block SARS-CoV-2 replication specifically. We next encapsulated 2NSP23 nanobody into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in cells and suppresses multiple SARS-CoV-2 variants as seen by qPCR and RNA deep sequencing. These results are corroborated in 3D reconstituted human epithelium kept at air/liquid interface to mimic the outer surface of lung tissue. These observations indicate that LNP-mRNA-2NSP23 is internalized and following translation, it inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative strategy to generate novel antiviral drugs highly efficient across coronaviruses.

Employing Machine Learning-Based QSAR for Targeting Zika Virus NS3 Protease: Molecular Insights and Inhibitor Discovery.

Zika virus infection is a mosquito-borne viral disease that has become a global health concern recently. Zika virus belongs to the Flavivirus genus and is primarily transmitted by Aedes mosquitoes. Prevention of Zika virus infection involves avoiding mosquito bites by using repellent, wearing protective clothing, and staying in screened areas, especially for pregnant women. Treatment focuses on managing symptoms with rest, fluids, and acetaminophen, with close monitoring for pregnant women. Currently, there is no specific antiviral treatment or vaccine for the Zika virus, highlighting the importance of prevention strategies to control its spread. Therefore, in this study, the Zika virus non-structural protein NS3 was targeted to inhibit Zika infection by identifying the novel inhibitor through an in silico approach. Here, 2864 natural compounds were screened using a machine learning-based QSAR model, and later docking was performed to select the potential target. Subsequently, Tanimoto similarity and clustering were performed to obtain the potential target. The three most potential compounds were obtained: (a) 5297, (b) 432449, and (c) 85137543. The protein-ligand complex's stability and flexibility were then investigated by dynamic modelling. The 300 ns simulation showed that 5297 exhibited the steadiest deviation and constant creation of hydrogen bonds. Compared to the other compounds, 5297 demonstrated a superior binding free energy (ΔG = -20.81 kcal/mol) with the protein when the MM/GBSA technique was used. The study determined that 5297 showed significant therapeutic potential and justifies further experimental investigation as a possible inhibitor of the NS2B-NS3 protease target implicated in Zika virus infection.

Molecular architecture of coronavirus double-membrane vesicle pore complex

Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications1,2. SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs3–5. The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3–nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3–nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection.

Dengue virus: pathogenesis and potential for small molecule inhibitors.

Dengue, caused by dengue virus (DENV), is now endemic in nearly 100 countries and infection incidence is reported in another 30 countries. Yearly an estimated 400 million cases and 2200 deaths are reported. Effective vaccines against DENV are limited and there has been significant focus on the development of effective antiviral against the disease. The World Health Organization has initiated research programs to prioritize the development and optimization of antiviral agents against several viruses including Flaviviridae. A significant effort has been taken by the researchers to develop effective antivirals against DENV. Several potential small-molecule inhibitors like efavirenz, tipranavir and dasabuvir have been tested against envelope and non-structural proteins of DENV, and are in clinical trials around the world. We recently developed one small molecule, namely 7D, targeting the host PF4-CXCR3 axis. 7D inhibited all 4 serotypes of DENV in vitro and specifically DENV2 infection in two different mice models. Although the development of dengue vaccines remains a high priority, antibody cross reactivity among the serotypes and resulting antibody-dependent enhancement (ADE) of infection are major concerns that have limited the development of effective vaccine against DENV. Therefore, there has been a significant emphasis on the development of antiviral drugs against dengue. This review article describes the rescue effects of some of the small molecule inhibitors to viral/host factors associated with DENV pathogenesis.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains.

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.

Computational Insights for Interactions between nsP2 and nsP3 of CHIKV and Hormones through DFT Computations and Molecular Dynamics Simulations.

The non-structural protein (nsP2 & nsP3) of the Chikungunya virus (CHIKV) is responsible for the transmission of viral infection. The main role of non-structural proteins are involved in the transcription process at an early stage of the infection. In this work, authors have studied the impact of nsP2 and nsP3 of CHIKV on hormones present in the human body using a computational approach. The ten hormones of chemical properties such as 4-Androsterone-2,17-dione, aldosterone, androsterone, corticosterone, cortisol, cortisone, estradiol, estrone, progesterone and testosterone were taken as a potency. From the molecular docking, the binding energy of the complexes is estimated, and cortisone was found to be the highest negative binding energy (-6.57 kcal/mol) with the nsP2 and corticosterone with the nsP3 (-6.47 kcal/mol). This is based on the interactions between hormones and nsP2/nsP3, which are types of noncovalent intermolecular interactions categorized into three types: electrostatic interactions, van der Waals (vdW) interactions, and hydrogen-bonding (H-bonding) interactions. To validate the docking results, additional molecular dynamics simulations and MM-GBSA methods were performed. The change in enthalpy, entropy, and free energy were calculated using MM-GBSA methods. The nsP2 and nsP3 of CHIKV interact strongly with the cortisone and corticosterone with free energy changes of -20.55 & -36.08 kcal/mol, respectively. Methods: The crystal structures of 3TKR and 3GPO proteins of nsP2 and nsP3 were extracted from the RCSB Protein Data Bank. Initially, unnecessary atoms like extra cations or anions and missing explicit hydrogen atoms were removed and added from the native domain of nsP2 and nsP3. The alignment of coordinated in the native domain was performed using Chimera and Notepad++ tools. The molecular docking of protein and ligand was performed usingAutoDock tool; it is essential for the prediction of the orientation of the ligand into the cavity of the target protein based on binding affinity. Based on thermodynamic parameters, MD Simulations were employed to calculate the change in binding free energies of various complexes followed by a change in enthalpy and entropy with time. According to MD production, the CPPTAJ and PTRAJ programs were used to analyse the trajectories, such as dynamic stability (RMSD), residual fluctuation (RMSF), compatibility, and hydrogen bonds of the newly formed complexes. After that, the Density Functional Theory (DFT) were used to calculate the electronic properties of selected molecules by Gaussian 16 on applying the B3LYP method with the 6-311G (d, p) basis set.

An Undetected Expansion, Spread, and Burden of Chikungunya and Dengue Cocirculating Antibodies in Nigeria

Chikungunya and dengue are arboviral diseases transmitted by mosquitoes that have been increasingly recognized as public health concerns in Sub-Saharan Africa. Several studies conducted in Nigeria and other West African countries have revealed the seroprevalence burden and cocirculation of antibodies against mosquito-borne infections, thereby revealing a significant burden and clinical outcome complexities that have largely gone undetected. The current research study has important implications for disease surveillance, prevention strategies, and healthcare planning in Nigeria and other Sub-Saharan countries. A cross-sectional study was conducted on 871 outpatients and pregnant women from three regions of Nigeria. CHIKV and DENV immunoblot molecular diagnostic assays were used to analyze the serum samples for the presence of arboviral antibody serological markers IgG (Mikrogen Diagnostik, Germany) with DENV nonstructural protein 1 and DENV Equad and CHIKV virus-like particles (VLPs), according to the manufacturer’s instructions. A total of 871 participants were recruited from three geographical regions in Nigeria. Among them, 17.5% (152/871) were from Abia (southern Nigeria), 34.4% (300/871) were from Kaduna (northern Nigeria), and 48.1% (419/871) were from Nasarawa (central Nigeria). The ages of the participants ranged from 0 months to 80 years, with a mean age of 36.6 years. Of the 871 subjects, 71.0% (619/871) were female, and 29.0% (252/871) were male. The overall cohort detectable antibody seropositivity against CHIKV was 64.9% (565/871), 95% CI (61.74–68.06); DENV, 44.7% (389/871), 95% CI (41.41–47.99); and CHIKV-DENV cocirculation antibodies, 31.6% (95% CI 29–35). This study highlighted the unpredictably high seroprevalence, expansion, magnitude, and undetected burden of chikungunya and dengue in Nigeria.

Insect ribosome-rescuer Pelo-Hbs1 complex on sperm surface mediates paternal arbovirus transmission

Arboviruses can be paternally transmitted by male insects to offspring for long-term persistence, but the mechanism remains largely unknown. Here, we use a model system of a destructive rice reovirus and its leafhopper vector to find that insect ribosome-rescuer Pelo-Hbs1 complex expressed on the sperm surface mediates paternal arbovirus transmission. This occurs through targeting virus-containing tubules constituted by viral nonstructural protein Pns11 to sperm surface via Pns11-Pelo interaction. Tubule assembly is dependent on Hsp70 activity, while Pelo-Hbs1 complex inhibits tubule assembly via suppressing Hsp70 activity. However, virus-activated ubiquitin ligase E3 mediates Pelo ubiquitinated degradation, synergistically causing Hbs1 degradation. Importantly, Pns11 effectively competes with Pelo for binding to E3, thus antagonizing E3-mediated Pelo-Hbs1 degradation. These processes cause a slight reduction of Pelo-Hbs1 complex in infected testes, promoting effective tubule assembly. Our findings provide insight into how insect sperm-specific Pelo-Hbs1 complex is modulated to promote paternal virus transmission without disrupting sperm function.

Tick-borne encephalitis vaccine breakthrough infections induce aberrant T cell and antibody responses to non-structural proteins

Tick-borne encephalitis virus (TBEV) vaccine breakthrough (VBT) infections are not uncommon in endemic areas. The clinical and immunological outcomes have been poorly investigated. We assessed the magnitude and specificity of virus-specific antibody and T cell responses after TBE in previously vaccinated subjects and compared the results with those of unvaccinated TBE patients and study subjects that received vaccination without VBT infection. Symptomatic TBEV infection of unvaccinated study subjects induced virus-specific antibody responses to the E protein and non-structural protein 1 (NS1) as well as T cell responses to structural and other non-structural (NS) proteins. After VBT infections, significantly impaired NS1-specific antibody responses were observed, while the virus-specific T cell responses to the NS proteins were relatively strong. VBT infection caused predominantly moderate to severe disease during hospitalization. The level of TBEV EDIII- and NS1-specific antibodies in unvaccinated convalescent patients inversely correlated with TBE severity and neurological symptoms early after infection.

Exploring T-Cell Immunity to Hepatitis C Virus: Insights from Different Vaccine and Antigen Presentation Strategies.

The hepatitis C virus (HCV) is responsible for approximately 50 million infections worldwide. Effective drug treatments while available face access barriers, and vaccine development is hampered by viral hypervariability and immune evasion mechanisms. The CD4+ and CD8+ T-cell responses targeting HCV non-structural (NS) proteins have shown a role in the viral clearance. In this paper, we reviewed the studies exploring the relationship between HCV structural and NS proteins and their effects in contributing to the elicitation of an effective T-cell immune response. The use of different vaccine platforms, such as viral vectors and virus-like particles, underscores their versability and efficacy for vaccine development. Diverse HCV antigens demonstrated immunogenicity, eliciting a robust immune response, positioning them as promising vaccine candidates for protein/peptide-, DNA-, or RNA-based vaccines. Moreover, adjuvant selection plays a pivotal role in modulating the immune response. This review emphasizes the importance of HCV proteins and vaccination strategies in vaccine development. In particular, the NS proteins are the main focus, given their pivotal role in T-cell-mediated immunity and their sequence conservation, making them valuable vaccine targets.

Disrupting protease and deubiquitinase activities of SARS-CoV-2 papain-like protease by natural and synthetic products discovered through multiple computational and biochemical approaches

The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (−7.97 kcal/mol to −8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 μM and deubiquitinase activity = 3.11 ± 0.75 μM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 μM in the human BJ cell line.

Viral methyltransferase inhibitors: berbamine, venetoclax, and ponatinib as efficacious antivirals against chikungunya virus

Chikungunya virus (CHIKV), transmitted by mosquitoes, poses a significant global health threat. Presently, no effective treatment options are available to reduce the disease burden. The lack of approved therapeutics against CHIKV and the complex spectrum of chronic musculoskeletal and neurological manifestations raise significant concerns, and repurposing drugs could offer swift avenues in the development of effective treatment strategies. RNA capping is a crucial step meditated by non-structural protein 1 (nsP1) in CHIKV replication. In this study, FDA-approved antivirals targeting CHIKV nsP1 methyltransferase (MTase) have been identified by structure-based virtual screening. Berbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) were the top-hits, which exhibited robust binding energies. Tryptophan fluorescence spectroscopy-based assay confirmed binding of BH-, ABT-, and PT to purified nsP1 with KD values ∼5.45 μM, ∼161.3 μM, and ∼3.83 μM, respectively. In a capillary electrophoresis-based assay, a decrease in CHIKV nsP1 MTase activity was observed in a dose-dependent manner. Treatment with BH, ABT, and PT lead to a dose-dependent reduction in the virus titer with IC50 < 100, ∼6.75, and <3.9 nM, respectively, and reduced viral mRNA levels. The nsP1 MTases are highly conserved among alphaviruses; therefore, BH, ABT, and PT, as expected, inhibited replication machinery in Sindbis virus (SINV) replicon assay with IC50 ∼1.94, ∼0.23, and >1.25 μM, respectively. These results highlight the potential of repurposing drugs as rapid and effective antiviral therapeutics against CHIKV.

Subsequent Waves of Convergent Evolution in SARS-CoV-2 Genes and Proteins.

Beginning in 2022, following widespread infection and vaccination among the global population, the SARS-CoV-2 virus mainly evolved to evade immunity derived from vaccines and past infections. This review covers the convergent evolution of structural, nonstructural, and accessory proteins in SARS-CoV-2, with a specific look at common mutations found in long-lasting infections that hint at the virus potentially reverting to an enteric sarbecovirus type.

3-chymotrypsin-like protease in SARS-CoV-2.

Coronaviruses constitute a significant threat to the human population. Severe acute respiratory syndrome coronavirus-2, SARS-CoV-2, is a highly pathogenic human coronavirus that has caused the coronavirus disease 2019 (COVID-19) pandemic. It has led to a global viral outbreak with an exceptional spread and a high death toll, highlighting the need for effective antiviral strategies. 3-Chymotrypsin-like protease (3CLpro), the main protease in SARS-CoV-2, plays an indispensable role in the SARS-CoV-2 viral life cycle by cleaving the viral polyprotein to produce 11 individual non-structural proteins necessary for viral replication. 3CLpro is one of two proteases that function to produce new viral particles. It is a highly conserved cysteine protease with identical structural folds in all known human coronaviruses. Inhibitors binding with high affinity to 3CLpro will prevent the cleavage of viral polyproteins, thus impeding viral replication. Multiple strategies have been implemented to screen for inhibitors against 3CLpro, including peptide-like and small molecule inhibitors that covalently and non-covalently bind the active site, respectively. In addition, allosteric sites of 3CLpro have been identified to screen for small molecules that could make non-competitive inhibitors of 3CLpro. In essence, this review serves as a comprehensive guide to understanding the structural intricacies and functional dynamics of 3CLpro, emphasizing key findings that elucidate its role as the main protease of SARS-CoV-2. Notably, the review is a critical resource in recognizing the advancements in identifying and developing 3CLpro inhibitors as effective antiviral strategies against COVID-19, some of which are already approved for clinical use in COVID-19 patients.

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.

The Flavivirus Non-Structural Protein 5 (NS5): Structure, Functions, and Targeting for Development of Vaccines and Therapeutics.

Flaviviruses, including dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), yellow fever (YFV), and tick-borne encephalitis (TBEV) viruses, pose a significant global emerging threat. With their potential to cause widespread outbreaks and severe health complications, the development of effective vaccines and antiviral therapeutics is imperative. The flaviviral non-structural protein 5 (NS5) is a highly conserved and multifunctional protein that is crucial for viral replication, and the NS5 protein of many flaviviruses has been shown to be a potent inhibitor of interferon (IFN) signalling. In this review, we discuss the functions of NS5, diverse NS5-mediated strategies adopted by flaviviruses to evade the host antiviral response, and how NS5 can be a target for the development of vaccines and antiviral therapeutics.

Unlocking Secrets: Lipid Metabolism and Lipid Droplet Crucial Roles in SARS-CoV-2 Infection and the Immune Response.

Lipid droplets (LD) are crucial for maintaining lipid and energy homeostasis within cells. LDs are highly dynamic organelles that present a phospholipid monolayer rich in neutral lipids. Additionally, LDs are associated with structural and non-structural proteins, rapidly mobilizing lipids for various biological processes. Lipids play a pivotal role during viral infection, participating during viral membrane fusion, viral replication, and assembly, endocytosis, and exocytosis. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection often induces LD accumulation, which is used as a source of energy for the replicative process. These findings suggest that LDs are a hallmark of viral infection, including SARS-CoV-2 infection. Moreover, LD participates in the inflammatory process and cell signaling, activating pathways related to innate immunity and cell death. Accumulating evidence demonstrates that LD induction by SARS-CoV-2 is a highly coordinated process, aiding replication and evading the immune system, and may contribute to the different cell death process observed in various studies. Nevertheless, recent research in the field of LDs suggests these organelles according to the pathogen and infection conditions may also play roles in immune and inflammatory responses, protecting the host against viral infection. Understanding how SARS-CoV-2 influences LD biogenesis is crucial for developing novel drugs or repurposing existing ones. By targeting host lipid metabolic pathways exploited by the virus, it is possible to impact viral replication and inflammatory responses. This review seeks to discuss and analyze the role of LDs during SARS-CoV-2 infection, specifically emphasizing their involvement in viral replication and the inflammatory response.

Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain

The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures. Currently, the mechanism underlying its attenuation phenotype remain largely unknown. In this study, we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques. The recovered H2 strain (H2ic) from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production, similar to the parental H2 strain. Additionally, H2ic did not cause disease in Ifnar1−/− C57 mice, consistent with the H2 strain. To explore the cell-adaptive mutations of the H2 strain, chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone. The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic. Other chimeric viruses containing the 2B, 2C, or 3A proteins from H2w failed to be recovered. Furthermore, there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses. These results demonstrate that adaptive mutations in the 2B, 2C, and 3A proteins are essential for efficient replication of the H2 strain in cell cultures. Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice. Together, this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.

Computational Investigations to Identify Potent Natural Flavonoid Inhibitors of the Nonstructural Protein (NSP) 16/10 Complex Against Coronavirus.

Globally, the viral pandemic has spread rapidly, resulting in widespread infections. The coronavirus family (CoVs) is one of the various viral families capable of infecting mammals, causing diseases related to the gastrointestinal, neurological, and respiratory systems. Flavonoid compounds have been identified as potentially effective antiviral agents, specifically targeting the virus's nonstructural protein (NSP) 16/10. Flavonoids have also been shown to inhibit virus replication and viral attachment to host cells, making them a promising candidate for antiviral treatment. Further research is needed to understand the full potential of flavonoids as antiviral agents. This study investigated natural compounds derived from medicinal plants using in silico screening. In addition to assessing drug-likeness, pharmacokinetics, docking, molecular dynamics simulation, bioavailability assessment, and exploration of molecular targets, the screening process entailed analyses of molecular targets and bioavailability. The molecular properties and potential antiviral efficacy of these phytochemical candidates were determined by analyzing them as drug candidates. The results of the study showed that these compounds had potential antiviral activity and could be developed as therapeutic agents. Furthermore, the study showed that the compounds had good bioavailability, suggesting that they are suitable for use as therapeutic agents. An in silico method was used to identify flavonoid compounds for potent antiviral drug molecules against the coronavirus protein complex NSP16/10 protein. The NSP16/10 complex protein binding energy values were -6.14 for isoquercetin, -6.902 for narirutin, -6.052 for myricetin, -7.10 for hesperidin, -4.392 for silibinin, -3.997 for baicalein, -3.712 for taxifolin, and -3.321 for petunidin. Molecular dynamics simulations showed that isoquercetin, hesperidin, and narirutin flavonoids interacted with the COVID-19 virus protein complex NSP16/10 protease up to 100 nanoseconds.