Viral Life Cycle Research Articles

Identification and characterization of host factor VCPIP1 as a multi-functional positive regulator of hepatitis B virus.

Hepatitis B virus (HBV) encodes the regulatory protein HBx that plays crucial roles in viral life cycle and cellular processes through interacting with viral and cellular proteins. Identifying HBx-interacting proteins may reveal novel aspects of host-virus interactions. In this work, proximity labeling coupled with proteomic analysis identified multiple HBx-interacting host factors, and among these, valosin-containing protein-interacting protein 1 (VCPIP1) was confirmed to directly bind HBx and reduce its proteasomal degradation, corroborating a recent report. In addition to upregulating HBx-expressing HBV, we showed that VCPIP1 also positively regulates mutant HBV lacking HBx expression. This novel HBx-independent function of VCPIP1 was shown to involve its association with one viral promoter/enhancer element, which upregulated the latter's promoter and enhancer activities. These results establish VCPIP1 as a positive regulator of HBV that acts through multiple, diverse mechanisms and might also contribute toward revealing novel cellular functions of VCPIP1.

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.

BK Polyomavirus Infection of Bladder Microvascular Endothelial Cells Leads to the Activation of the cGAS-STING Pathway.

BK polyomavirus (BKPyV) infection in humans is usually asymptomatic but ultimately results in viral persistence. In immunocompromised hosts, virus reactivation can lead to nephropathy or hemorrhagic cystitis. The urinary tract serves as a silent reservoir for the virus. Recently, it has been demonstrated that human bladder microvascular endothelial cells (HBMVECs) serve as viral reservoirs, given their unique response to infection, which involves interferon (IFN) production. The aim of the present study was to better understand the life cycle of BKPyV in HBMVECs, uncover the molecular pathway leading to IFN production, and to identify the connection between the viral life cycle and the activation of the IFN response. Here, in the early stage of infection, BKPyV virions were found in internalized monopinocytic vesicles, while later they were detected in late endosomes, lysosomes, tubuloreticular structures, and vacuole-like vesicles. The production of viral progeny in these cells started at 36 h postinfection. Increased cell membrane permeability and peaks of virion release coincided with the leakage of viral and cellular DNA into the cytosol at approximately 60 h postinfection. Leaked DNA colocalized with and activated cGAS, leading to the activation of STING and the consequent transcription of IFNB and IFN-related genes; in contrast, the IFN response was attenuated by exposure to the cGAS inhibitor, G140. These findings highlight the importance of the cGAS-STING pathway in the innate immune response of HBMVECs to BKPyV.

Viral proteins resolve the virus-vector conundrum during hemipteran-mediated transmission by subverting salicylic acid signaling pathway

Hemipteran insects transmit viruses when infesting plants, during which vectors activate salicylic acid (SA)-regulated antiviral defenses. How vector-borne plant viruses circumvent these antiviral defenses is largely unexplored. During co-infections of begomoviruses and betasatellites in plants, betasatellite-encoded βC1 proteins interfere with SA signaling and reduce the activation of antiviral resistance. βC1 inhibits SA-induced degradation of NbNPR3 (Nicotiana benthamiana nonexpressor of pathogenesis-related genes 3), a negative regulator of SA signaling. βC1 does not bind directly to NbNPR3, but regulates NbNPR3 degradation via heat shock protein 90s (NbHSP90s). NbHSP90s bind to both NbNPR3 and βC1 and suppress SA signaling. This viral success strategy appears to be conserved as it is also documented for viral proteins encoded by two aphid-borne viruses. Our findings reveal an exquisite mechanism that facilitates the persistence of vector-borne plant viruses and provide important insights into the intricacies of the virus life cycle.

Host-Driven Ubiquitination Events in Vector-Transmitted RNA Virus Infections as Options for Broad-Spectrum Therapeutic Intervention Strategies

Many vector-borne viruses are re-emerging as public health threats, yet our understanding of the virus–host interactions critical for productive infection remains limited. The ubiquitination of proteins, including host- and pathogen-derived proteins is a highly prominent and consistent post-translational modification that regulates protein function through signaling and degradation. Viral proteins are documented to hijack the host ubiquitination machinery to modulate multiple host processes including antiviral defense mechanisms. The engagement of the host ubiquitination machinery in the post-translational modification of viral proteins to support aspects of the viral life cycle including assembly and egress is also well documented. Exploring the role ubiquitination plays in the life cycle of vector-transmitted viral pathogens will increase the knowledge base pertinent to the impact of host-enabled ubiquitination of viral and host proteins and the consequences on viral pathogenesis. In this review, we explore E3 ligase-regulated ubiquitination pathways functioning as proviral and viral restriction factors in the context of acutely infectious, vector-transmitted viral pathogens and the potential for therapeutically targeting them for countermeasures development.

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Mitochondrial Dysfunction and Metabolic Disturbances Induced by Viral Infections

Viruses are intracellular parasites that utilize organelles, signaling pathways, and the bioenergetics machinery of the cell to replicate the genome and synthesize proteins to build up new viral particles. Mitochondria are key to supporting the virus life cycle by sustaining energy production, metabolism, and synthesis of macromolecules. Mitochondria also contribute to the antiviral innate immune response. Here, we describe the different mechanisms involved in virus–mitochondria interactions. We analyze the effects of viral infections on the metabolism of glucose in the Warburg phenotype, glutamine, and fatty acids. We also describe how viruses directly regulate mitochondrial function through modulation of the activity of the electron transport chain, the generation of reactive oxygen species, the balance between fission and fusion, and the regulation of voltage-dependent anion channels. In addition, we discuss the evasion strategies used to avoid mitochondrial-associated mechanisms that inhibit viral replication. Overall, this review aims to provide a comprehensive view of how viruses modulate mitochondrial function to maintain their replicative capabilities.

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 proline-rich 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 development novel attenuated vaccines against PRRSV.

Immunoreactivity Analysis of MHC-I Epitopes Derived from the Nucleocapsid Protein of SARS-CoV-2 via Computation and Vaccination

Background: Since 2019, the SARS-CoV-2 virus has been responsible for the global spread of respiratory illness. As of 1 September 2024, the cumulative number of infections worldwide exceeded 776 million. There are many structural proteins of the virus, among which the SARS-CoV-2 nucleocapsid (N) protein plays a pivotal role in the viral life cycle, participating in a multitude of essential activities following viral invasion. An important antiviral immune response is the major histocompatibility complex (MHC)-restricted differentiation cluster 8 (CD8+) T cell cytotoxicity. Therefore, understanding the immunogenicity of SARS-CoV-2 NP-specific MHC-I-restricted epitopes is highly important. Methods: MHC-I molecules from 11 human leukocyte antigen I (HLA-I) superfamilies with 98% population coverage and 6 mouse H2 alleles were selected. The affinity were screened by IEDB, NetMHCpan, SYFPEITHI, SMMPMBEC and Rankpep. Further immunogenicity and conservative analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis was performed. Selective epitopes were validated by enzyme-linked immunospot (ELISpot) assay and flow cytometry in the mice with pVAX-NPSARS-CoV-2 immunization. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect whether the preferred epitope induced humoral immunity. Results: There were 64 dominant epitopes for the H-2 haplotype and 238 dominant epitopes for the HLA-I haplotype. Further analysis of immunogenicity and conservation yielded 8 preferred epitopes, and docking simulations were conducted with corresponding MHC-I alleles. The relationships between the NP peptides and MHC-I haplotypes were then determined via two-way hierarchical clustering. ELISA, ELISpot assay, and flow cytometry revealed that the preferred epitope stimulated both humoral and cellular immunity and enhanced cytokine secretion in mice. Conclusions: our study revealed the general patterns among multiple haplotypes within the humans and mice superfamily, providing a comprehensive assessment of the pan-MHC-I immunoreactivity of SARS-CoV-2 NP. Our findings would render prospects for the development and application of epitope-based immunotherapy in lasting viral epidemics.

HBV biomarkers and their role in guiding treatment decisions.

Over 300 million individuals worldwide are chronically infected with Hepatitis B Virus and at risk for progressive liver disease. Due to the lack of a therapy that reliably achieves viral elimination and the variability of liver disease progression, treatment decisions are guided by the degree of liver disease and viral biomarkers as the viral life-cycle is well characterised and largely conserved between individuals. In contrast, the immunological landscape is much more heterogenous and diverse and the measurement of its components is less well standardized. Due to the lack of a universal and easily measurable set of biomarkers, clinical practice guidelines remain controversial, aiming for a balance between simplifying treatment decisions by reducing biomarker requirements and using all available biomarkers to avoid overtreatment of patients with low risk for disease progression. While approved therapies such as nucleos(t)ide analogues improve patient outcomes, the inability to achieve complete cure highlights the need for novel therapies. Since no treatment candidate has demonstrated universal efficacy, biomarkers will remain important for treatment stratification. Here, we summarize the current knowledge on virological and immunological biomarkers with a specific focus on how they might be beneficial in guiding treatment decisions in chronic Hepatitis B.

Insight into structural properties of viral G protein-coupled receptors and their role in the viral infection: IUPHAR Review 41.

G protein-coupled receptors (GPCRs) are pivotal in cellular signalling and drug targeting. Herpesviruses encode GPCRs (vGPCRs) to manipulate cellular signalling, thereby regulating various aspects of the virus life cycle, such as viral spreading and immune evasion.vGPCRs mimic host chemokine receptors, often with broader signalling and high constitutive activity. This review focuses on the recent advancements in structural knowledge about vGPCRs, with an emphasis on molecular mechanisms of action and ligand binding. The structures of US27 and US28 from human cytomegalovirus (HCMV) are compared to their closest human homologue, CX3CR1. Contrasting US27 and US28, the homotrimeric UL78 structure (HCMV) reveals more distance to chemokine receptors. Open reading frame 74 (ORF74; Kaposi's sarcoma-associated herpesvirus) is compared to CXCRs, whereas BILF1 (Epstein-Barr virus) is discussed as a putative lipid receptor. Furthermore, the rolesof vGPCRs in latency and lytic replication, reactivation, dissemination and immune evasion arereviewed, together with their potential as drug targets for virus infections and virus-related diseases.

Alternative splicing in the genome of HPV and its regulation.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer. These chronic infections are characterized by high expression of the HPV E6 and E7 oncogenes and the absence of the L1 and L2 capsid proteins. The regulation of HPV gene expression plays a crucial role in both the viral life cycle and rare oncogenic events. Alternative splicing of HPV mRNA is a key mechanism in post-transcriptional regulation. Through alternative splicing, HPV mRNA is diversified into various splice isoforms with distinct coding potentials, encoding multiple proteins and influencing the expression of HPV genes. The spliced mRNAs derived from a donor splicing site within the E6 ORF and one of the different acceptor sites located in the early mRNA contain E6 truncated mRNAs, named E6*. E6* is one of the extensively studied splicing isoforms. However, the role of E6* proteins in cancer progression remains controversial. Here, we reviewed and compared the alternative splicing events occurring in the genomes of HR-HPV and LR-HPV. Recently, new HPV alternative splicing regulatory proteins have been continuously discovered, and we have updated the regulation of HPV alternative splicing. In addition, we summarized the functions of known splice isoforms from three aspects: anti-tumorigenic, tumorigenic, and other cancer-related functions, including not only E6*, but also E6^E7, E8^E2, and so on. Comprehending their contributions to cancer development enhances insights into the carcinogenic mechanisms of HPV and explores the potential utility of alternative splicing in the diagnosis and treatment of cervical cancer.

A transcriptionally active lipid vesicle encloses the injected Chimalliviridae genome in early infection.

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses 1-3 . Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA 4-10 . Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae , but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART 11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

The Role of Peptides in Combatting HIV Infection: Applications and Insights.

Peptide-based inhibitors represent a promising approach for the treatment of HIV-1, offering a range of potential advantages, including specificity, low toxicity, and the ability to target various stages of the viral lifecycle. This review outlines the current state of research on peptide-based anti-HIV therapies, highlighting key advancements and identifying future research directions. Over the past few years, there has been significant progress in developing synthetic peptide-based drugs that target various stages of the viral life cycle, including entry and replication. These approaches aim to create effective anti-HIV therapies. Additionally, peptides have proven valuable in the development of anti-HIV vaccines. In the quest for effective HIV vaccines, discovering potent antigens and designing suitable vaccine strategies are crucial for overcoming challenges such as low immunogenicity, safety concerns, and increased viral load. Innovative strategies for vaccine development through peptide research are, therefore, a key focus area for achieving effective HIV prevention. This review aims to explore the strategies for designing peptides with anti-HIV activity and to highlight their role in advancing both therapeutic and preventive measures against HIV.

Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between Two Membrane-Associated Condensate States.

The SARS-CoV-2 Nucleocapsid protein (N) performs several functions during the viral lifecycle, including transcription regulation and viral genome encapsulation. We hypothesized that N toggles between these functions via phosphorylation-induced conformational change, thereby altering N interactions with membranes and RNA. We found that phosphorylation changes how biomolecular condensates composed of N and RNA interact with membranes: phosphorylated N (pN) condensates form thin films, while condensates with unmodified N are engulfed. This partly results from changes in material properties, with pN forming less viscous and elastic condensates. The weakening of protein-RNA interaction in condensates upon phosphorylation is driven by a decrease in binding between pN and unstructured RNA. We show that phosphorylation induces a conformational change in the serine/arginine-rich region of N that increases interaction between pN monomers and decreases nonspecific interaction with RNA. These findings connect the conformation, material properties, and membrane-associated states of N, with potential implications for COVID-19 treatment.

RNA architecture of porcine deltacoronavirus genome inside virions detected by vRIC-seq

Porcine deltacoronavirus (PDCoV) is a newly emerging and special delta coronavirus, which infect mammals such as pigs, cattle and humans, as well as chickens and birds. Exploring RNA structures in the viral genome benefits the understanding of the role of RNA in the lifecycle of viruses. In this study, vRIC-seq is employed to analyze the RNA-RNA interaction in the whole genome structure of PDCoV in virions. About 12.87 and 13.52 million paired reads are obtained in two biological replicates, respectively, with 17.9% and 14.8% of them are identified as valid chimeric reads. These are employed to predict the RNA secondary structure, which is compact and highly structured. A twisted-cyclized conformation is observed in the RNA-RNA interaction map of PDCoV for the first time. 77 multi-way junctions are evenly distributed in the PDCoV genome. Our work provides fundamental structural insights that are essential for understanding the genomic structure and function, genetic evolution, and packaging characteristics of PDCoV.

Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line.

Cells are equipped with intracellular RIG-like Receptors (RLRs) detecting double stranded (ds)RNA, a molecule with Pathogen-Associated Molecular Pattern (PAMPs) generated during the life cycle of many viruses. Melanoma Differentiation-Associated protein 5 (MDA5), a helicase enzyme member of the RLRs encoded by the ifih1 gene, binds to long dsRNA molecules during a viral infection and initiates production of type I interferon (IFN1) which orchestrates the antiviral response. In order to understand the contribution of MDA5 to viral resistance in fish cells, we have isolated a clonal Chinook salmon Oncorhynchus tshawytscha epithelial-like cell line invalidated for the ifih1 gene by CRISPR/Cas9 genome editing. We demonstrated that IFN1 induction is impaired in this cell line after infection with the Snakehead Rhabdovirus (SHRV), the Salmon Alphavirus (SAV) or Nervous Necrosis Virus (NNV). The cell line, however, did not show any increase in cytopathic effect when infected with SHRV or SAV. Similarly, no cytopathic effect was observed in the ifih1-/- cell line when infected with Infectious Pancreatic Necrosis Virus (IPNV), Infectious Haemorrhagic Necrotic Virus (IHNV). These results indicate the redundancy of the antiviral innate defence system in CHSE-derived cells, which helps with circumventing viral evasion strategies.

Targeting Human Papillomavirus 33 E2 DNA Binding Domain With Polyphenols: Unveiling Interactions Through Biophysical and In Silico Methods.

The human papillomavirus (HPV) 33 is a high-risk strain that causes lesions with potential cancerous outcomes. Its E2 protein regulates the viral protein transcription and life cycle maintenance. The DNA binding domain (DBD) of the E2 protein plays a crucial role in the viral life cycle. The DBD region of the E2 protein is particularly interesting for targeting and finding potential inhibitors to inhibit its function or dimerization. Given the limited research on HPV 33 and its proteins, the present work delved into the interaction of two natural polyphenolic compounds, resveratrol, and baicalein, with the E2 DBD of HPV 33 using biophysical and in silico studies. Fluorescence studies of the E2 DBD-polyphenol complexes showed fluorescence quenching with a binding constant of the order of 106 M-1. Circular dichroism data reveal conformational changes upon binding with the polyphenols, possibly due to distinct binding sites of the E2 DBD. Differential scanning calorimetry exhibited higher melting temperatures for the two complexes than alone DBD, suggesting the complexes' stability. ITC experiment suggested favorable binding reactions with kd values in the micromolar range. Molecular docking and dynamic simulation studies revealed that the resveratrol binds to the helical region and baicalein near the central dimeric interface of E2 DBD with a good binding affinity, forming a stable protein-ligand complex during the run of 100 ns simulation. Therefore, the current study identifies both polyphenolic compounds as promising candidates for potential antiviral drug development.

Efficacy of Integrase Strand Transfer Inhibitors and the Capsid Inhibitor Lenacapavir against HIV-2, and Exploring the Effect of Raltegravir on the Activity of SARS-CoV-2

Retroviruses perpetuate their survival by incorporating a copy of their genome into the host cell, a critical step catalyzed by the virally encoded integrase. The viral capsid plays an important role during the viral life cycle, including nuclear importation in the case of lentiviruses and integration targeting events; hence, targeting the integrase and the viral capsid is a favorable therapeutic strategy. While integrase strand transfer inhibitors (INSTIs) are recommended as first-line regimens given their high efficacy and tolerability, lenacapavir is the first capsid inhibitor and the newest addition to the HIV treatment arsenal. These inhibitors are however designed for treatment of HIV-1 infection, and their efficacy against HIV-2 remains widely understudied and inconclusive, supported only by a few limited phenotypic susceptibility studies. We therefore carried out inhibition profiling of a panel of second-generation INSTIs and lenacapavir against HIV-2 in cell culture, utilizing pseudovirion inhibition profiling assays. Our results show that the tested INSTIs and lenacapavir exerted excellent efficacy against ROD-based HIV-2 integrase. We further evaluated the efficacy of raltegravir and other INSTIs against different variants of SARS-CoV-2; however, contrary to previous in silico findings, the inhibitors did not demonstrate significant antiviral activity.

Characterization of duck tembusu virus NS2A membrane topology and functional residues in transmembrane domain-3 on viral proliferation

Flavivirus nonstructural protein 2A (NS2A) is a small endoplasmic reticulum (ER)-resident, hydrophobic transmembrane protein that function in viral replication, virion assembly and evasion of the host immune response. Despite previous studies on the role of duck Tembusu virus (DTMUV) NS2A in inhibiting the host immune response, its membrane topology has not been clearly addressed (Zhang et al., 2020; Zhang et al., 2022). Here, we present the first report on the membrane topology model and functional characterization of DTMUV NS2A. Our findings demonstrate that DTMUV NS2A localizes to the endoplasmic reticulum (ER) and associates with viral double-stranded RNA, with a single segment (TMD3, amino acids 72 to 95) spanning the ER membrane. To better delineate the residues in NS2A-TMD3 related to viral properties, specific mutations were introduced to generate DTMUV replicons and infectious cDNA clones. Functional analysis indicates that L77, Q86 and L89 of NS2A are crucial for viral RNA synthesis, while residues M79 and F83 are crucial for the assembly or release of viral particles. Moreover, these mutations attenuated the virulence of DTMUV in vivo. Collectively, our results shed light on the relationship between the transmembrane of DTMUV NS2A and its functions in virus proliferation, providing insights for further understanding the molecular mechanisms of NS2A in the virus life cycle.