USP26 as a hepatitis B virus-induced deubiquitinase primes hepatocellular carcinogenesis by epigenetic remodeling.

Despite recent advances in systemic therapy for hepatocellular carcinoma (HCC), the prognosis of hepatitis B virus (HBV)-induced HCC patients remains poor. By screening a sgRNA library targeting human deubiquitinases, we find that ubiquitin-specific peptidase 26 (USP26) deficiency impairs HBV-positive HCC cell proliferation. Genetically engineered murine models with Usp26 knockout confirm that Usp26 drives HCC tumorigenesis. Mechanistically, we find that the HBV-encoded protein HBx binds to the promoter and induces the production of USP26, which is an X-linked gene exclusively expressed in the testis. HBx consequently promotes the association of USP26 with SIRT1 to synergistically stabilize SIRT1 by deubiquitination, which promotes cell proliferation and impedes cell apoptosis to accelerate HCC tumorigenesis. In patients with HBV-positive HCC, USP26 is robustly induced, and its levels correlate with SIRT1 levels and poor prognosis. Collectively, our study highlights a causative link between HBV infection, deubiquitinase induction and development of HCC, identifying a druggable target, USP26.

Hepatitis B Virus X Protein Induces Expression Changes of miR-21, miR-22, miR-122, miR-132, and miR-222 in Huh-7 Cell Line.

Hepatitis B virus (HBV) X protein (HBx) plays a key role in hepatocellular carcinoma (HCC). HBx may alter the expression of multiple microRNAs (miRs), which are important in hepatocarcinogenesis. This study aimed to investigate the importance of HBx protein in the expression of miR-21, miR-22, miR-122, miR-132, and miR-222. A recombinant vector expressing HBx was developed. The Huh-7 cell line was transfected with the HBx-pcDNA3.1+ recombinant plasmid. A Real-Time Polymerase Chain Reaction was used to evaluate the expression of miR-21, miR-22, miR-122, miR-132, and miR-222 in the cell line. It was found that the expression of miR-21 and miR-222 was upregulated at all points of time after HBx transfection. The expression of miR-21 was 4.24-fold 72 h after transfection. The miR-22 had a 7.69-fold downregulation after 24 h, and the miR-122 had a significant downregulation after 48 h (10-fold). The miR-132 expression reached its lowest rate 12 h after HBx transfection (8.33-fold), and the miR-222 expression was upregulated in transfected cells but was not significantly different (1.18- to 2.45-fold). The significant downregulation of miR-22, miR-122, and miR-132 implicates their inhibitory roles in the progression of HBV-associated HCC. The expression of these microRNAs could be used as a prognostic marker for the progression of HBV-associated liver disease.

CRL4-DCAF1 Ubiquitin Ligase Dependent Functions of HIV Viral Protein R and Viral Protein X.

The Human Immunodeficiency Virus (HIV) encodes several proteins that contort the host cell environment to promote viral replication and spread. This is often accomplished through the hijacking of cellular ubiquitin ligases. These reprogrammed complexes initiate or enhance the ubiquitination of cellular proteins that may otherwise act to restrain viral replication. Ubiquitination of target proteins may alter protein function or initiate proteasome-dependent destruction. HIV Viral Protein R (Vpr) and the related HIV-2 Viral Protein X (Vpx), engage the CRL4-DCAF1 ubiquitin ligase complex to target numerous cellular proteins. In this review we describe the CRL4-DCAF1 ubiquitin ligase complex and its interactions with HIV Vpr and Vpx. We additionally summarize the cellular proteins targeted by this association as well as the observed or hypothesized impact on HIV.

NTB-A and 2B4 Natural Killer Cell Receptors Modulate the Capacity of a Cocktail of Non-Neutralizing Antibodies and a Small CD4-Mimetic to Eliminate HIV-1-Infected Cells by Antibody-Dependent Cellular Cytotoxicity.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

SIGIRR suppresses hepatitis B virus X protein-induced chronic inflammation in hepatocytes.

Although antiviral drugs can effectively inhibit hepatitis B virus (HBV) replication, the maintenance of chronic inflammation in the liver is still considered to be an important cause for the progression of HBV-related liver disease to liver fibrosis and advanced liver disease. As an endogenous inhibitory receptor of IL-1R and TLR signaling pathways, single immunoglobulin interleukin-1-related receptor (SIGIRR) has been proven to reduce inflammation in tissues to maintain system homeostasis. However, the relationship between SIGIRR expression and HBV replication and inflammatory pathway activation in hepatocytes remains unclear. In this study, hepatitis B virus X protein (HBx) upregulated MyD88 in liver cells, promoting NF-κB signaling and inflammatory factor production with LPS treatment, and the cell supernatant accelerated the activation and collagen secretion of hepatic stellate cells. However, SIGIRR overexpression suppressed HBx-mediated MyD88/NF-κB inflammatory signaling activation and inflammatory cytokine production induced by LPS in hepatocytes and HBV replication hepatocytes. Although we did not find any effect of SIGIRR on HBV replication in vitro, this study investigated the role of SIGIRR in blocking the proinflammatory function of HBx, which may provide a new idea for the treatment of chronic hepatitis B.

SOD1 inhibition enhances sorafenib efficacy in HBV-related hepatocellular carcinoma by modulating PI3K/Akt/mTOR pathway and ROS-mediated cell death.

Hepatitis B Virus (HBV) infection significantly elevates the risk of hepatocellular carcinoma (HCC), with the HBV X protein (HBx) playing a crucial role in cancer progression. Sorafenib, the primary therapy for advanced HCC, shows limited effectiveness in HBV-infected patients due to HBx-related resistance. Numerous studies have explored combination therapies to overcome this resistance. Sodium diethyldithiocarbamate (DDC), known for its anticancer effects and its inhibition of superoxide dismutase 1 (SOD1), is hypothesized to counteract sorafenib (SF) resistance in HBV-positive HCCs. Our research demonstrates that combining DDC with SF significantly reduces HBx and SOD1 expressions in HBV-positive HCC cells and human tissues. This combination therapy disrupts the PI3K/Akt/mTOR signalling pathway and promotes apoptosis by increasing reactive oxygen species (ROS) levels. These cellular changes lead to reduced tumour viability and enhanced sensitivity to SF, as evidenced by the synergistic suppression of tumour growth in xenograft models. Additionally, DDC-mediated suppression of SOD1 further enhances SF sensitivity in HBV-positive HCC cells and xenografted animals, thereby inhibiting cancer progression more effectively. These findings suggest that the DDC-SF combination could serve as a promising strategy for overcoming SF resistance in HBV-related HCC, potentially optimizing therapy outcomes.

Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2.

A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2'-O-methyltransferase (2'-O-MTase), to cap their RNAs through ribose 2'-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2'-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2'-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2'-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2'-O methylation process and mechanism, providing valuable targets for antiviral drug development.

In silico exploration of deep-sea fungal metabolites as inhibitor of Ebola and Marburg VP35 and VP40.

VP30 and VP40 proteins of Ebola and Marburg viruses have been recognized as potential targets for antiviral drug development due to their essential roles in the viral lifecycle. Targeting these proteins could disrupt key stages of the viral replication process, inhibiting the viruses' ability to propagate and cause disease. The current study aims to perform molecular docking and virtual screening on deep-sea fungal metabolites targeting Marburg virus VP40 Dimer, matrix protein VP40 from Ebola virus Sudan, Ebola VP35 Interferon Inhibitory Domain, and VP35 from Marburg virus. The top ten compounds for each protein target were chosen using the glide score. All the compounds obtained indicate a positive binding interaction. Furthermore, AdmetSAR was utilized to investigate the pharmacokinetics of the inhibitors chosen. Gliotoxin was used as a ligand with Marburg virus VP40 Dimer, Austinol with matrix protein VP40 from Ebola virus Sudan, Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) with Ebola VP35 Interferon Inhibitory Domain, and Dehydroaustinol with VP35 from Marburg virus. MD modeling and MMPBSA studies were used to provide a better understanding of binding behaviors. Pre-clinical experiments can assist validate our in-silico studies and assess whether the molecule can be employed as an anti-viral drug.

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10.

Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

HBx promotes glomerular podocyte-induced immune cell responses.

BACKGROUND: Podocytes, as intrinsic renal cells, can also express MHC-II and costimulatory molecules under inflammatory conditions, suggesting that they may act as antigen-presenting cells (APCs) to activate immune cell responses and then lead to immune-mediated renal injury. They are already recognized as main targets in the pathogenic mechanism of hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN). Previous studies also have indicated that inflammatory cells infiltration and immune-mediated tissue injury are evident in the kidney samples of patients with HBV-GN. However, the role of podocytes immune disorder in the pathogenic mechanism of HBV-GN remains unclear. METHODS: Renal function and inflammatory cells infiltration were measured in HBV transgenic (HBV-Tg) mice. In vitro, podocytes/CD4+ T cells or macrophages co-culture system was established. Then, the expression of HBx, CD4, and CD68 was determined by immunohistochemistry, while the expression of MHC-II, CD40, and CD40L was determined by immunofluorescence. Co-stimulatory molecules expression was examined by flow cytometry. The levels of inflammatory factors were detected by ELISA. RESULTS: In vivo, renal function was obviously impaired in HBV-Tg mice. HBx was significantly upregulated and immune cells infiltrated in the glomerulus of HBV-Tg mice. Expression of MHC-II and costimulatory molecule CD40 increased in the podocytes of HBV-Tg mice; CD4+ T cells exhibited increased CD40L expression in glomerulus. In vitro, CD40 expression was markedly elevated in HBx-podocytes. In co-culture systems, HBx-podocytes stimulated CD4+ T cells activation and caused the imbalance between IFN-γ and IL-4. HBx-podocytes also enhanced the adhesion ability of macrophages and induced the release of proinflammatory mediators. CONCLUSION: Taken together, these podocyte-related immune disorder may be involved in the pathogenic mechanism of HBV-GN.

Novel Vpx virus-like particles to improve cytarabine treatment response against acute myeloid leukemia.

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.

High level of genomic divergence in orf-I p12 and hbz genes of HTLV-1 subtype-C in Central Australia.

BACKGROUND: Human T cell lymphotropic virus type 1 (HTLV-1) infection remains a largely neglected public health problem, particularly in resource-poor areas with high burden of communicable and non-communicable diseases, such as some remote populations in Central Australia where an estimated 37% of adults are infected with HTLV-1. Most of our understanding of HTLV-1 infection comes from studies of the globally spread subtype-A (HTLV-1a), with few molecular studies reported with the Austral-Melanesian subtype-C (HTLV-1c) predominant in the Indo-Pacific and Oceania regions. RESULTS: Using a primer walking strategy and direct sequencing, we constructed HTLV-1c genomic consensus sequences from 22 First Nations participants living with HTLV-1c in Central Australia. Phylogenetic and pairwise analysis of this subtype-C proviral gDNA showed higher levels of genomic divergence in comparison to previously published HTLV-1a genomes. While the overall genomic homology between subtypes was 92.5%, the lowest nucleotide and amino acid sequence identity occurred near the 3' end of the proviral genome coding regulatory genes, especially overlapping hbz (85.37%, 77.46%, respectively) and orf-I product p12 (82.00%, 70.30%, respectively). Strikingly, the HTLV-1c genomic consensus sequences uniformly showed a defective translation start codon for the immune regulatory proteins p12/p8 encoded by the HTLV-1A orf-I. Deletions in the proviral genome were detected in many subjects, particularly in the structural gag, pol and env genes. Similarly, using a droplet digital PCR assay measuring the copies of gag and tax per reference host genome, we quantitatively confirmed that provirus retains the tax gene region at higher levels than gag. CONCLUSIONS: Our genomic analysis of HTLV-1c in Central Australia in conjunction with earlier Melanesian HTLV-1c sequences, elucidate substantial differences with respect to the globally spread HTLV-1a. Future studies should address the impact these genomic differences have on infection and the regionally distinctive frequency of associated pulmonary disease. Understanding the host and virus subtype factors which contribute to the differential morbidity observed, is crucial for the development of much needed therapeutics and vaccine strategies against this highly endemic infection in remote First Nations communities in Central Australia.

The nuclear matrix protein HNRNPU restricts hepatitis B virus transcription by promoting OAS3-based activation of host innate immunity.

Heterogeneous nuclear protein U (HNRNPU) plays a pivotal role in innate immunity by facilitating chromatin opening to activate immune genes during host defense against viral infection. However, the mechanism by which HNRNPU is involved in Hepatitis B virus (HBV) transcription regulation through mediating antiviral immunity remains unknown. Our study revealed a significant decrease in HNRNPU levels during HBV transcription, which depends on HBx-DDB1-mediated degradation. Overexpression of HNRNPU suppressed HBV transcription, while its knockdown effectively promoted viral transcription, indicating HNRNPU as a novel host restriction factor for HBV transcription. Mechanistically, HNRNPU inhibits HBV transcription by activating innate immunity through primarily the positive regulation of the interferon-stimulating factor 2'-5'-oligoadenylate synthetase 3, which mediates an ribonuclease L-dependent mechanism to enhance innate immune responses. This study offers new insights into the host immune regulation of HBV transcription and proposes potential targets for therapeutic intervention against HBV infection.

Epstein-Barr virus deubiquitinating enzyme BPLF1 is involved in EBV carcinogenesis by affecting cellular genomic stability.

Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.

SIAH2 suppresses c-JUN pathway by promoting the polyubiquitination and degradation of HBx in hepatocellular carcinoma.

As an important protein encoded by hepatitis B virus (HBV), HBV X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). It has been shown that seven in absentia homologue 1 (SIAH1) could regulates the degradation of HBx through the ubiquitin-proteasome pathway. However, as a member of SIAH family, the regulatory effects of SIAH2 on HBx remain unclear. In this study, we first confirmed that SIAH2 could reduce the protein levels of HBx depending on its E3 ligase activity. Moreover, SIAH2 interacted with HBx and induced its K48-linked polyubiquitination and proteasomal degradation. Furthermore, we provided evidence that SIAH2 inhibits HBx-associated HCC cells proliferation by regulating HBx. In conclusion, our study identified a novel role for SIAH2 in promoting HBx degradation and SIAH2 exerts an inhibitory effect in the proliferation of HBx-associated HCC through inducing the degradation of HBx. Our study provides a new idea for the targeted degradation of HBx and may have great huge significance into providing novel evidence for the targeted therapy of HBV-infected HCC.

Identification of Prospective Ebola Virus VP35 and VP40 Protein Inhibitors from Myxobacterial Natural Products.

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.

Reactive Oxygen Species Induction by Hepatitis B Virus: Implications for Viral Replication in p53-Positive Human Hepatoma Cells.

Hepatitis B virus (HBV) infects approximately 300 million people worldwide, causing chronic infections. The HBV X protein (HBx) is crucial for viral replication and induces reactive oxygen species (ROS), leading to cellular damage. This study explores the relationship between HBx-induced ROS, p53 activation, and HBV replication. Using HepG2 and Hep3B cell lines that express the HBV receptor NTCP, we compared ROS generation and HBV replication relative to p53 status. Results indicated that HBV infection significantly increased ROS levels in p53-positive HepG2-NTCP cells compared to p53-deficient Hep3B-NTCP cells. Knockdown of p53 reduced ROS levels and enhanced HBV replication in HepG2-NTCP cells, whereas p53 overexpression increased ROS and inhibited HBV replication in Hep3B-NTCP cells. The ROS scavenger N-acetyl-L-cysteine (NAC) reversed these effects. The study also found that ROS-induced degradation of the HBx is mediated by the E3 ligase Siah-1, which is activated by p53. Mutations in p53 or inhibition of its transcriptional activity prevented ROS-mediated HBx degradation and HBV inhibition. These findings reveal a p53-dependent negative feedback loop where HBx-induced ROS increases p53 levels, leading to Siah-1-mediated HBx degradation and HBV replication inhibition. This study offers insights into the molecular mechanisms of HBV replication and identifies potential therapeutic targets involving ROS and p53 pathways.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Involvement of SARS-CoV-2 accessory proteins in immunopathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the largest single-stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end, recombinant SARS-CoV-2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10) was de novo generated using an early pandemic SARS-CoV-2 strain as a backbone. We confirmed that the resultant virus (termed ORF3-10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3-10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3-10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3-10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS-CoV-2 accessory proteins are involved in immunopathogenicity.

Hemodialysis bilayer bionic blood vessels developed by the mechanical stimulation of hepatitis B viral X(HBX) gene- transfected hepatic stellate cells. / é€šè¿‡æœºæ¢°åŠ›åˆºæ¿€ä¹™åž‹è‚ç‚Žç— æ¯’XåŸºå› (HBX)转染的肝星状细胞制备血液透析用双层仿生血管.

Artificial vascular graft (AVG) fistula is widely used for hemodialysis treatment in patients with renal failure. However, it has poor elasticity and compliance, leading to stenosis and thrombosis. The ideal artificial blood vessel for dialysis should replicate the structure and components of a real artery, which is primarily maintained by collagen in the extracellular matrix (ECM) of arterial cells. Studies have revealed that in hepatitis B virus (HBV)-induced liver fibrosis, hepatic stellate cells (HSCs) become hyperactive and produce excessive ECM fibers. Furthermore, mechanical stimulation can encourage ECM secretion and remodeling of a fiber structure. Based on the above factors, we transfected HSCs with the hepatitis B viral X (HBX) gene for simulating the process of HBV infection. Subsequently, these HBX-HSCs were implanted into a polycaprolactone-polyurethane (PCL-PU) bilayer scaffold in which the inner layer is dense and the outer layer consists of pores, which was mechanically stimulated to promote the secretion of collagen nanofiber from the HBX-HSCs and to facilitate crosslinking with the scaffold. We obtained an ECM-PCL-PU composite bionic blood vessel that could act as access for dialysis after decellularization. Then, the vessel scaffold was implanted into a rabbit's neck arteriovenous fistula model. It exhibited strong tensile strength and smooth blood flow and formed autologous blood vessels in the rabbit's body. Our study demonstrates the use of human cells to create biomimetic dialysis blood vessels, providing a novel approach for creating clinical vascular access for dialysis.