Viral Life Cycle Research Articles

Establishment of minigenomes for infectious bursal disease virus

Minigenomes (MGs) have greatly advanced research on the viral life cycle, including viral replication and transcription, virus‒host interactions, and the discovery of antivirals against RNA viruses. However, an MG for infectious bursal disease virus (IBDV) has not been well established. Here, we describe the development of IBDV MG, in which the entire coding sequences of viral genomic segments A and B are replaced with Renilla luciferase (Rluc) or enhanced green fluorescent protein (EGFP) reporter genes. Under the control of the RNA polymerase I promoter, the translation of IBDV MG is controlled by the viral proteins VP1 and VP3. Interestingly, IBDV B MG shows greater activity than does IBDV A MG. Moreover, the sense IBDV B MG was expressed at a higher level than the antisense IBDV B MG. In agreement with our previous findings, the translation of IBDV B MG controlled by VP1 and VP3 is independent of the cellular translation machinery components eukaryotic initiation factor (eIF)4E and eIF4G, but intact VP1 polymerase activity, VP3 dsRNA-binding activity, and the interaction between VP1 and VP3 are indispensable for both sense and antisense IBDV B MG activity. In addition, ribavirin, which inhibits IBDV replication, inhibits IBDV B MG activity in a dose-dependent manner. Collectively, the IBDV MG established in this study provides a powerful tool to investigate IBDV intracellular replication and transcription and virus‒host interactions and facilitates high-throughput screening for the identification of IBDV antivirals.

Potential role of Mappain, a prenylated stilbene, in reducing SARS-CoV-2 envelope protein-induced inflammation

As of right now, more than 776 million people have contracted the COVID-19-causing SARS-CoV-2 virus, which has also resulted in over 7 million deaths. A dysregulated host's innate immune response to the virus exacerbates clinical outcomes and leads to cytokine storm. Developing a drug targeting the multiple aspects of the SARS-CoV-2 virus-host cell interactions is essential for effective treatment. The 75 amino acid-long SARS-CoV-2 envelope (2E) protein is a small structural transmembrane protein that plays a crucial role in the viral life cycle and pathogenesis. Mappain is a plant-derived stilbene from Macaranga species that previously demonstrated antiviral potential against enteroviruses in tissue culture. This study aims to assess the inhibitory potential of Mappain against SARS-CoV-2 key proteins and induced hyperinflammation using computational and in-vitro approaches. Autodock Vina was used to evaluate mappain interaction with SARS- CoV-2 antiviral targets including Spike, Envelope, Membrane, 3CLPro, PLpro, Nucleocapsid N and C termini, RdRP, and host receptors—TLR2, TLR3, TLR7, and TLR8. Afterwards, mappain was subjected to in-vitro bioassay testing using inhibition 2E-induced cytokines (IL-6 and TNF-α) in human peripheral blood mononuclear cells, measured by ELISA; Amantadine as control. KEGG pathway enrichment analysis was further performed using network pharmacology to elucidate the mechanism. Mappain demonstrated a high binding score with all the targets, especially with the envelope protein (− 9.6 kcal/mol) and TLR2 (− 9.7 kcal/mol) compared to Amantadine (− 6.1 kcal/mol and − 5.5 kcal/mol, respectively). Mappain also significantly reduced the expression of 2E-induced Interleukin 6 (p ≤ 0.001) and tumour necrosis factor-alpha (p ≤ 0.01) in the PBMCs, when compared to Amantadine HCl. The KEGG Pathway enrichment analysis supports the anti-inflammatory potential of mappain in the virus-induced cytokine storm by specifically targeting TLR signalling and COVID-19 pathways.

In-depth analysis of serum antibodies against Epstein-Barr virus lifecycle proteins, and EBNA1, ANO2, GlialCAM and CRYAB peptides in patients with multiple sclerosis

BackgroundA strong association between multiple sclerosis (MS) and Epstein-Barr virus (EBV) has been established but the exact role of EBV in MS remains controversial. Recently, molecular mimicry between EBNA1 and specific GlialCAM, CRYAB and ANO2 peptides has been suggested as a possible pathophysiological mechanism. The aim of this study was to analyse anti-EBV antibodies in MS patients against (I) EBV lifecycle proteins, (II) putative cross-reactive peptides, and (III) during treatment.MethodsIn this retrospective cross-sectional study, 258 serum samples were included consisting of EBV-negative (n = 25) and EBV-positive (n = 36) controls, 192 MS samples including untreated relapsing-remitting MS (RRMS) with and without relapses, secondary progressive MS (SPMS) and primary progressive MS (PPMS) patients, and 106 patients on 8 different treatment regimens. IgG and IgM antibody titers against EBV docking/fusion proteins (gp350, gh/gp42, gh/gL/gp42), immediate early antigen (BZLF1), early antigens (EA p85, EA P138, EA P54), capsid antigens (VCA P18, VCA P23, VCA gp125) and late antigens (EBNA1) were measured. Specific EBNA1 and GlialCAM, CRYAB and ANO2 peptides were synthesized and also incorporated in our custom magnetic bead based multiplex assay.ResultsWe observed significantly elevated IgG antibody titers in EBV-positive controls, RRMS with and without relapse, SPMS and PPMS patients for all lifecycle antigens except for several early antigens when compared to EBV-negative controls. Significantly higher IgG antibody titers were observed in RRMS patients for fusion proteins and EBNA1 peptides when compared to EBV-positive controls. An MS specific response was observed for ANO2 but not for GlialCAM or CRYAB. No significant treatment effects or a specific IgM response were detectable.ConclusionThe MS-specific, differential antibody response to EBV antigens confirms an altered immunological response to EBV in MS patients. EBV reactivation does not appear to play an important role in MS pathogenesis and no differential antibody signatures were observed between MS disease phases. The MS-specific anti-ANO2 antibody response suggests a potential role for EBNA1 as an antigenic driver, although the exact role of anti-ANO2 antibodies needs to be determined. The precise pathophysiological role of EBV in MS remains uncertain and requires further investigation.

Molecular sociology of virus-induced cellular condensates supporting reovirus assembly and replication

Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication.

Evaluations of FDA-approved Drugs Targeting 3CLP of SARS-CoV-2 Employing a Repurposing Strategy.

The SARS-CoV-2 coronavirus (COVID-19) has raised innumerable global concerns, and few effective treatment strategies have yet been permitted by the FDA to lighten the disease burden. SARS-CoV-2 3C-like proteinase (3CLP) is a crucial protease and plays a key role in the viral life cycle, as it controls replication, and thus, it is viewed as a target for drug design. In this study, we performed structure-based virtual screening of FDA drugs approved during 2015-2019 (a total of 220 drugs) for interaction with the active site of 3CLP (PDB ID 6LU7) using AutoDock 4.2. We report the top ten drugs that outperform the reported drugs against 3CLP (Elbasvir and Nelfinavir), particularly Cefiderocol, having the highest affinity among the compounds tested, with a binding energy of -9.97 kcal/mol. H-bond (LYS102:HZ2-ligand: O49), hydrophobic (ligand-VAL104), and electrostatic (LYS102:NZ-ligand: O50) interactions were observed in the cefiderocol-3CLP complex. The docked complex was subjected to a 50 ns molecular dynamics study to check its stability, and stable RMSD and RMSF graphs were observed. Accordingly, we suggest cefiderocol might be effective against SARS-CoV-2 and urge that experimental validation be performed to determine the antiviral efficacy of cefiderocol against SARS-CoV-2. Along with these, cefiderocol is effective for treating respiratory tract pathogens and a wide range of gram-negative bacteria for whom there are limited therapeutic alternatives. This article aimed to explore the FDA-approved drugs as a repurposing study against 3CLP for COVID-19 management.

Hepatitis C Virus—Core Antigen: Implications in Diagnostic, Treatment Monitoring and Clinical Outcomes

The hepatitis C virus (HCV) infection, a global health concern, can lead to chronic liver disease. The HCV core antigen (HCVcAg), a viral protein essential for replication, offers a cost-effective alternative to HCV RNA testing, particularly in resource-limited settings. This review explores the significance of HCVcAg, a key protein in the hepatitis C virus, examining its structure, function, and role in the viral life cycle. It also evaluates its clinical use in diagnosis and treatment monitoring, comparing its performance to the standard HCV RNA assay using data from PubMed and Google Scholar. HCVcAg assays show high pooled sensitivity (93.5%) and pooled specificity (99.2%) compared to HCV RNA assays, correlating closely (r = 0.87) with HCV RNA levels. Hence, HCVcAg testing offers a cost-effective way to diagnose active HCV infections and monitor treatment, especially in resource-limited settings, but its sensitivity can vary and standardization is needed. HCVcAg also predicts liver disease progression and assesses liver damage risk, aiding patient management. It helps to identify patients at risk for fibrosis or carcinoma, making it vital in hepatitis C care. HCVcAg testing can expand access to HCV care, simplify management, and contribute to global elimination strategies, especially in low- and middle-income countries.

The characterization and structural basis of a human broadly binding antibody to HBV core protein.

Hepatitis B virus (HBV) core protein (HBc) plays a crucial role in the virus life cycle, making it an important detection marker for HBV infection and a potential target for treatment. However, several commercially available monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) targeting HBc have certain limitations in detecting HBV across different genotypes in various biochemical assays, such as enzyme-linked immunosorbent assay, western blot, immunofluorescence assay, flow cytometry, and immune spot assay. In this study, we identified 12 human anti-HBc mAbs and evaluated their potential application in multiple biochemical assays. These mAbs mainly recognized the epitopes near residues 20-22 amino acids (a.a.) and 77-78 a.a. of HBc, demonstrating a broadly cross-genotypic activity. Notably, three Group II mAbs, named cAbA1, cAbD4, and cAbF9, displayed excellent capacities for the detection of HBV infection in all tested biochemical assays. Furthermore, we determined a 3.22 Å of cryo-electron microscopy (cryo-EM) structure of the fragment of antigen binding (Fab) of cAbD4 complexed with HBc dimer, which was the highest resolution of the structural model for Fab-HBc to date. Collectively, our findings provided excellent and reliable antibody candidates for live HBV detection and revealed the recognition mechanism and the detailed interaction information of a potent human anti-HBc mAb binding to the spike tips of HBc dimer.IMPORTANCEThe lack of excellent detection Abs for live hepatitis B virus (HBV) infection and high-resolution structures of the Ab-HBV core protein (HBc) complex largely limited the development of HBV-related research. This study reports a panel of anti-HBc monoclonal antibodies (mAbs) with excellent capacities for detecting HBV infection in multiple biochemical assays and determines a 3.22 Å of cryo-EM structure of HBc with a potent binding mAb. These findings provide excellent and reliable detecting tools for HBV-related research and promote the understanding of the recognition mechanism of anti-HBc mAbs to HBc particles.

Three-dimensional liver organoids as a novel platform for studying hepatitis B virus infection

Hepatitis B virus (HBV) infection is a major global health problem that contributes to chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. Despite advances in antiviral therapy and the availability of preventive vaccines, current treatments often do not completely eradicate the virus, particularly in chronic HBV carriers, and the incidence of liver cancer due to chronic HBV infection remains high. A major obstacle to HBV research is the lack of infection models that can accurately recapitulate the complex interactions between HBV and the host. Existing models, such as hepatoma cell lines and animal models, are limited by species-specific barriers and the lack of support for the complete viral life cycle. These limitations pose significant challenges to the study of HBV pathogenesis and the development of therapies aimed at complete eradication of HBV. In recent years, 3-dimensional (3D) liver organoids have emerged as promising in vitro model systems to study HBV infection and HBV-mediated liver disease. These organoids serve as a suitable physiologically relevant platform for investigating HBV pathogenesis, including the ability of HBV to promote liver tumorigenesis. Furthermore, liver organoids can be genetically modified, patient-derived, expanded, and biobanked, providing a powerful tool for studying HBV pathogenesis and personalized medicine, drug discovery, and screening. In this review, we explore the utilization of 3D liver organoids as a research model for studying HBV infection and HBV-associated liver cancer, highlighting their advantages over conventional models.

Discovery of orally bioavailable SARS-CoV-2 papain-like protease inhibitor as a potential treatment for COVID-19

The RNA-dependent RNA polymerase (RdRp), 3C-like protease (3CLpro), and papain-like protease (PLpro) are pivotal components in the viral life cycle of SARS-CoV-2, presenting as promising therapeutic targets. Currently, all FDA-approved antiviral drugs against SARS-CoV-2 are RdRp or 3CLpro inhibitors. However, the mutations causing drug resistance have been observed in RdRp and 3CLpro from SARS-CoV-2, which makes it necessary to develop antivirals with novel mechanisms. Through the application of a structure-based drug design (SBDD) approach, we discover a series of novel potent non-covalent PLpro inhibitors with remarkable in vitro potency and in vivo PK properties. The co-crystal structures of PLpro with lead compounds reveal that the residues D164 and Q269 around the S2 site are critical for improving the inhibitor’s potency. The lead compound GZNL-P36 not only inhibits SARS-CoV-2 and its variants at the cellular level with EC50 ranging from 58.2 nM to 306.2 nM, but also inhibits HCoV-NL63 and HCoV-229E with EC50 of 81.6 nM and 2.66 μM, respectively. Oral administration of the GZNL-P36 results in significantly improved survival and notable reductions in lung viral loads and lesions in SARS-CoV-2 infection mouse model, consistent with RNA-seq data analysis. Our results indicate that PLpro inhibitors represent a promising SARS-CoV-2 therapy.

Ultrasensitive Chemiluminescence Probes Designed from Covalent Inhibitors for SRAS-CoV-2 Mpro Detection.

In the postpandemic era, the emergence of "long COVID" from SARS-CoV-2 has brought ongoing negative impacts on individual health and society. The development of more efficient methods for drug screening and monitoring viral activity remains a critical need. The main protease (Mpro), due to its important role in the virus lifecycle, high conservation, and specificity, is considered an ideal biomarker for SARS-CoV-2. Herein, we have developed several chemiluminescence probes based on different substrates modified from covalent inhibitors targeted at Mpro. Among these, the best probe, MPCL-2, exhibits a rapid response (<20 min), an extremely low limit of detection (LoD; 0.11 nM), great selectivity, and chemical stability. After validating the probe's mechanism of action, MPCL-2 can also be used for real-time, in-situ imaging of enzymes in cells infected with the authentic virus and has the potential for real-time, in-situ Mpro imaging in vivo. Compared to other methods reported to date, the probe demonstrates superior performance and broader applicability, such as in drug screening or virus activity monitoring. Further, the unique design strategy for the substrate can be adopted to develop sensitive probes for other pathogens.

Distinct epigenetic and transcriptional profiles of Epstein-Barr virus (EBV) positive and negative primary CNS lymphomas.

Epstein-Barr virus (EBV)+ and EBV- primary CNS lymphomas (PCNSL) carry distinct mutational landscapes, but their transcriptional and epigenetic profiles have not been integrated and compared. This precludes further insights into pathobiology and molecular differences, relevant for classification and targeted therapy. 23 EBV- and 15 EBV+ PCNSL, histologically classified as diffuse large B-cell lymphomas, were subjected to RNA-Sequencing and EPIC methylation arrays. Unsupervised clustering analyses were performed. Differentially expressed and differentially methylated genes were identified and integrated. Two distinct transcriptional clusters were found, which separated EBV-and EBV+PCNSL (p < 0.0001). The EBV+ transcriptional signature contained genes (GPR15, FCER2/CD23, SLAMF1/CD150) closely regulated by EBV oncogenes in B-cells. Pathway enrichment analysis uncovered enhanced B-cell receptor (BCR) and WNT/beta-catenin signaling in EBV-lymphomas, whereas Interleukin-10, NOTCH, and viral life cycle pathways were upregulated in EBV+PCNSL. Correspondingly, BCR-associated SYK kinase activity was enriched in EBV-tumors while JAK2 was overrepresented in EBV+PCNSL. Epigenetic profiling revealed reduced global promoter methylation in EBV+PCNSL. Two methylation clusters were recognized, which separated EBV-and EBV+PCNSL (p < 0.0001). Epigenetic profiles were distinct from 2,788 other brain tumor and non-malignant reference samples. Promoter region hypermethylation of CD79B, a BCR subunit critical for sustained proliferation in EBV-disease, highly correlated (R = -0.7) with its transcriptional downregulation in EBV+PCNSL. EBV+ and EBV- PCNSL harbor distinct transcriptional and epigenetic profiles, corroborating them as distinctive biological subtypes. Uncovered differences provide novel insights into their pathobiology, may guide molecular diagnostics and targeted therapies.

Identification of NECTIN1 as a novel restriction factor for flavivirus infection.

NECTIN1, also known as CD111 or PVRL1, has been recognized as the primary receptor for several alpha herpesviruses, including herpes simplex virus (HSV), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). However, our study revealed a novel role for NECTIN1 in the virus life cycle by influencing BVDV infection. Contrary to its role as a receptor for alpha herpesviruses, NECTIN1 acts as a restriction factor for BVDV by inhibiting viral attachment via competition with CD46 for binding to the domain DD of BVDV E2. We further revealed that the replication of members of the Flaviviridae family was inhibited by NECTIN1, while the replication of other RNA viruses did not significantly differ. Our results demonstrate that NECTIN1 is a novel factor restricting Flaviviridae family virus replication and highlight the complexity of virus-host interactions and the multifaceted nature of host factors involved in viral infection.

Insights into the role of N6-methyladenosine (m6A) in plant-virus interactions.

N6-methyladenosine (m6A) is a common and dynamic epitranscriptomic modification in eukaryotic RNAs, affecting stability, splicing, translation, and degradation. Recent technological advancements have revealed the complex nature of m6A modifications, highlighting their importance in plant and animal species. The m6A modification is a reversible process, with "writers" depositing methylation, "erasers" demethylating it, and "reader" proteins recognizing m6A and executing various biological functions. Studying the relationship between m6A methylation and viral infection is crucial. Animal viruses, including retroviruses, RNA viruses, and DNA viruses, often employ the host's m6A machinery to replicate or avoid immune responses. In plant viruses, host methyltransferases or demethylases can stabilize or degrade viral RNA, depending on the virus-host interaction. Additionally, viral infections can modify the host's m6A machinery, impacting the viral life cycle. This review examines the role of m6A modifications in plant viral pathogenesis, focussing on RNA viruses infecting crops like alfalfa, turnip, wheat, rice, and potato. Understanding the role of m6A in virus-host interactions can aid in studying plant viral disease development and discovering novel antiviral targets for crop protection. In this review, we summarize current information on m6A in RNA biology, focussing on its function in viral infections and plant-virus interactions.

RNA Modifications in Pathogenic Viruses: Existence, Mechanism, and Impacts.

RNA modification is a key posttranscriptional process playing various biological roles, and one which has been reported to exist extensively in cellular RNAs. Interestingly, recent studies have shown that viral RNAs also contain a variety of RNA modifications, which are regulated dynamically by host modification machinery and play critical roles in different stages of the viral life cycle. In this review, we summarize the reports of four typical modifications reported on viral RNAs, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N4-acetylcytosine (ac4C), and N1-methyladenosine (m1A), describe the molecular mechanisms of these modification processes, and illustrate their impacts on viral replication, pathogenicity, and innate immune responses. Notably, we find that RNA modifications in different viruses share some common features and mechanisms in their generation, regulation, and function, highlighting the potential for viral RNA modifications and the related host machinery to serve as the targets or bases for the development of antiviral therapeutics and vaccines.

Analysis of the abundance and diversity of RNA secondary structure elements in RNA viruses using the RNAsselem Python package

Recent advancements in experimental and computational methods for RNA secondary structure detection have revealed the crucial role of RNA structural elements in diverse molecular processes within living cells. It has been demonstrated that the secondary structure of the entire viral genome is often responsible for performing crucial functions in the viral life cycle and also influences virus evolution. To investigate the role of viral RNA secondary structure, alongside experimental techniques, the use of bioinformatics tools is important for analyzing various secondary structure patterns, including hairpin loops, internal loops, multifurcations, external loops, bulges, stems, and pseudoknots. Here, we have introduced a Python package for analyzing RNA secondary structure elements in viral genomes, which includes the recognition of common secondary structure patterns, the generation of descriptive statistics for these structural elements, and the provision of their basic properties. We applied the developed package to analyze the secondary structures of complete viral genomes collected from the literature, aiming to gain insights into viral function and evolution. Both the package and the collection of secondary structures of viral genomes are available at http://github.com/KazanovLab/RNAsselem.

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 characterized and largely conserved between individuals. In contrast, the immunological landscape is much more heterogeneous 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 analogs improve patient outcomes, the inability to achieve a 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.

Inhibition of protein kinase D and its substrate phosphatidylinositol-4 kinase III beta blocks common human coronavirus replication.

Human coronaviruses can lead to a range of clinical symptoms, from asymptomatic infection to severe illness and death, with a limited array of antiviral drugs available. Protein kinase D (PKD) is involved in various cellular processes, such as cell proliferation, apoptosis, and membrane fission of the Golgi apparatus. However, the specific role of PKD in the human coronavirus life cycle remains unclear. In this study, we found that PKD inhibitors effectively attenuated human coronavirus replication at the trans-Golgi network (TGN) stage in the viral life cycle. Furthermore, inhibiting PKD reduced PI4KIIIβ activation, thereby blocking viral replication in the host cells. Importantly, PI4KIIIβ inhibitors also blocked human coronavirus replication. Thus, PKD may represent a promising therapeutic target against both current circulating and future emerging coronaviruses.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

Stabilization of a single-stranded DNA of adeno-associated virus by inverted terminal repeats

Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends.

In vitro characteristics of purified recombinant hepatitis C virus core protein

In our previous study, we established a method for purifying bacterially expressed HCV core 1–164 under non-denaturing conditions. In the present study, we elucidated the characteristics of the purified core. The purified HCV core exhibited a notable affinity for HCV RNA, with a Kd of 3 nM, as determined by a filter binding assay. Electron microscopy analysis revealed that the purified HCV core self-assembled with RNA into spherical structures approximately 50 nm in diameter. Additionally, we demonstrated the direct binding of the purified HCV core to the purified endoplasmic reticulum (ER). Moreover, lipid strip assays revealed specific binding of the purified HCV core to specific phospholipids, suggesting that the core plays a role in specific ER membrane domains. These studies reveal the biochemical characteristics of the HCV core that significantly advance our understanding of its role as a capsid protein in the viral lifecycle and pathogenesis.