Hepatitis B Virus X protein-mediated transcription of covalently closed circular DNA and its inhibition by blocking neddylation

Das Hepatitis B Virus (HBV) kann zu chronischer Leberinfektion führen und ist ein Risikofaktor für die Entstehung von Leberzirrhose und dem hepato-zellulären Karzinom. Für die Persistenz des Virus ist die Bildung und die Aufrechterhaltung der kovalent geschlossenen zirkulären DNS („covalently closed circular“ (ccc)DNS) zwingend notwendig, da sie als episomale Matrize für die Transkription im Kern der infizierten Hepatozyten dient. Die an cccDNS gebundene virale Transkription hängt vom HBV X Protein (HBx) sowie den durch HBx initiierten Abbau des wirtsspezifischen Restriktionsfaktors „structural maintenance of chromosome 5/6“ (SMC5/6) ab. Eine Grundvoraussetzung für die Ubiquitinierung und den Abbau von SMC 5/6 ist, dass HBx das Protein “DNA damage-binding protein1“ (DDB1) bindet und somit den Cullin 4A Ringligasekomplex rekrutiert, welcher für seine Aktivierung neddyliert werden muss. Nachdem eine verlässliche quantitative PCR etabliert wurde konnte die Kinetik der cccDNS Bildung in vier verschiedenen in vitro Infektionsmodellen sowie der Einfluss von Medikamenten (Interferon-α, Nukleos(t)id-Analoga,Inhibitoren des viralen Zelleintritts(„entry Inhibitor“) und Kapsid-Inhibitoren) auf die cccDNS untersucht werden. Im Vergleich zum Replikationsintermediat der gelockerten zirkulären („relaxed circular“; (rc)DNS) sind die Kopienzahlen der cccDNS in infizierten Hepatozyten niedrig (1-6 Kopien/infizierter Zelle). Der „entry Inhibitor“ Myrcludex B unterband effizient die (ccc)DNS Bildung. Hohe Interferon-α Dosen verringerten die cccDNS Menge während Nukleos(t)id-Analoga diese nicht vermindern konnten. Die Gabe von Kapsid Inhibitoren während der Infektionsphase, aber nicht zu einem späteren Zeitpunkt, führte zu einer Verringerung der cccDNS Menge. Indem wir HBx negative Virionen für die Infektion verwendet haben, konnten wir die Schlüsselrolle von HBx in der Kontrolle der cccDNS Transkription verifizieren. Wird HBx transient von einem authentischen Promotor exprimiert befindet es sich im Zellkern. Mit Hilfe eines lentiviral basierten Transkomplementationsansatzes, in dem die Transkription von HBx defizienten Virionen auf das Level von Wildtypviren gehoben wurde, konnten Schlüsselfragmente und -bereiche des HBx bestimmt werden. Der gesamte C-Terminus von HBx (Aminosäuren 51-154) war sowohl ausreichend als auch unentbehrlich um die Replikation HBx-defizienter Viren wieder herzustellen während der N-Terminus (Aminosäuren 1-50) keine transaktivierende Funktion aufzeigte. Zusätzlich waren zwei kürzere Fragmente (Aminosäure 51-142 und 58-142) teilweise funktional. Bemerkenswerterweise wies die HBx Mutante(R96E), welche nur noch eine geringe Bindeaktivität zu DDB1 aufweist, überhaupt keine Transaktivierungsaktivität mehr auf, was darauf hindeutet, dass die Bildung des HBx-DDB1 Komplexes für die maximal mögliche Transkription der cccDNS nötig ist. Des Weiteren untersuchten wir ob eine Hemmung der Neddylierung die Transkription erschwert. MLN4924, ein spezifischer Inhibitor des NEDD8-aktivierenden Enzyms 1 zeigte ein bemerkenswertes antivirales Potenzial. MLN4924 verringerte die HBV Transkription und virale Antigenexpression wirksam in nanomolarer Dosierung. Nach der Bildung der cccDNS unterdrückte das Medikament hochgradig die Transkription selbiger ohne dabei die cccDNS Menge zu beeinflussen. Die Unterbrechung der MLN4924 Gabe führte in HepaRGNTCP Zellen nicht zu einer Reaktivierung der cccDNS Transkription, wohingegen in HepG2NTCP Zellen eine schnelle Erholung von HBV beobachtet wurde. Bemerkenswerter Weise wurde die Transkription der cccDNS HBx-defizienter Viren nicht signifikant beeinfluss, was darauf schließen lässt, dass der MLN4924 Effekt auf das Wildtypvirus HBx-abhängig ist. MLN4924 verringert dabei selektiv die Transkription aller HBV Promotoren im Kontext der cccDNS, jedoch nicht bei integrierter viraler DNA. MLN4924 verhinderte der Abbau von SMC6,förderte dessen Wiederherstellung und unterband somit die Transkription der cccDNS. Zusammenfassen lässt sich sagen, dass nukleäres HBx DDB1 bindet, den Abbau von SMC6 induziert und so die Transkription der cccDNS fördert. Ein Angriff auf die Neddylierung unterbindet die Transkription der cccDNS und stellt die SMC6-abhängige Restriktion der cccDNS wieder her. Somit friert eine MLN4924 Behandlung die cccDNS in einem Zustand der „transkriptionellen Stille“ ein. Kleine Moleküle, die auf den HBx-DDB1-Cullin Komplex zielen könnten somit eine Vorausschau auf die nächstmögliche Therapie für HBV sein

Giant Hall Switching by Surface-State-Mediated Spin-Orbit Torque in a Hard Ferromagnetic Topological Insulator

Topological insulators (TI) can apply highly efficient spin-orbit torque (SOT) and manipulate the magnetization with their unique topological surface states, and their magnetic counterparts, magnetic topological insulators (MTI) offer magnetization without shunting and are one of the highest in SOT efficiency. Here, we demonstrate efficient SOT switching of a hard MTI, V-doped (Bi,Sb)2Te3 (VBST) with a large coercive field that can prevent the influence of an external magnetic field and a small magnetization to minimize stray field. A giant switched anomalous Hall resistance of 9.2 $k\Omega$ is realized, among the largest of all SOT systems. The SOT switching current density can be reduced to $2.8\times10^5 A/cm^2$, and the switching ratio can be enhanced to 60%. Moreover, as the Fermi level is moved away from the Dirac point by both gate and composition tuning, VBST exhibits a transition from edge-state-mediated to surface-state-mediated transport, thus enhancing the SOT effective field to $1.56\pm 0.12 T/ (10^6 A/cm^2)$ and the spin Hall angle to $23.2\pm 1.8$ at 5 K. The findings establish VBST as an extraordinary candidate for energy-efficient magnetic memory devices

Distinct Regulation of Host Responses by ERK and JNK MAP Kinases in Swine Macrophages Infected with Pandemic (H1N1) 2009 Influenza Virus

Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2′5′-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models

Strategies to Inhibit Hepatitis B Virus at the Transcript Level

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics

Evolutionary and Mutational Characterization of the First H5N8 Subtype Influenza A Virus in Humans

Highly pathogenic influenza A virus H5 subtype remains a risk for transmission in humans. The H5N8 subtype has caused multiple outbreaks in poultry in Europe over the past few winters. During one recent outbreak in poultry in Astrakhan, workers on the farm were also infected. So far, little is known about how this virus evolves and adapts to infect humans. Here, we performed a time-resolved phylogenetic analysis of 129 HA sequences representing all 1891 available H5N8 viruses collected from 2010 to 2020. We also conducted a whole-genome scan on the human virus at the protein level. We found that H5N8 viruses have spilled over in 34 European countries during the flu season of 2020–2021. These viruses underwent two significant evolutionary steps during 2015–2016 and after 2018. Furthermore, we characterized a number of critical mutations in all viral proteins except PB1-F2, which contribute to increased virulence and avian-to-human adaptation. Our findings suggested that the accumulated mutations under evolution led to quantitative and qualitative changes, likely allowing the virus to spread to humans. Given that the H5N8 virus is co-circulating with other H5 viruses in Europe, the risk of a pandemic should not be underestimated. Continental surveillance and pandemic preparedness are to be established

Reassortment and adaptive mutations of an emerging avian influenza virus H7N4 subtype in China.

Human infections with avian influenza viruses including H5, H7 and H9 hemagglutinin subtypes occur at a low rate. Among human infections with H7 viruses, regional outbreaks with H7N2, H7N3, H7N7 and H7N9 have been documented. Early in 2018, a human infection with a novel H7N4 avian influenza virus was reported in Jiangsu, China. This study is aimed at understanding the probable origin and molecular features of this emerging H7N4 virus. Genomic segments encoding hemagglutinin (HA) and neuraminidase (NA) of H7Nx and HxN4 viruses were compared with this H7N4 strain by alignment and phylogenetic tree analysis. Phylogenetic analysis indicated that the human H7N4 virus probably originated from multiple reassortments of avian H7N7 and H8N4 viruses for its HA and NA, respectively, and likely a regional uncharacterized virus for its internal segments. Our data excluded that circulating avian H9N2 viruses were the origin of the H7N4 internal segments, unlike the human H5N1 and H7N9 viruses that both had H9N2 backbones. This index case provided a unique opportunity to examine viral mutations by directly comparing the human isolate with its closest viral relatives isolated from avian species from the patient's farm, which may suggest critical mutations required for viral adaptation in humans. Whole-genome scanning was performed and the sequences of the human and related avian H7N4 isolates were compared. Mutations in PB2 (E627K), PB2 (K683T), PB1-F2 (N47S), HA (N283D), HA(K321E), NA(A137V), NA(K296R) and M2 (C19Y) were identified in the human isolate while no mutations were found in PB1, NP, NS1, and NS2 of the human H7N4 compared to the avian H7N4 viruses. Our data in this report provide further evidence for the genesis of this novel H7N4 virus with a multi-reassortment model and show molecular changes that might be responsible for the transmission of this virus from chickens or ducks to and subsequent replication in humans

Effect of Oral Tranexamic Acid on the Blood Transfusion Rate and the Incidence of Deep Vein Thromboembolism in Patients after TKA

Purpose. To explore the effect of oral tranexamic acid treatment on the blood transfusion rate and the incidence of deep vein thromboembolism after total knee arthroplasty (TKA). Methods. 90 patients undergoing TKA admitted to First People’s Hospital of Changshu City from January 2019 to January 2020 were selected and randomized into the control group and the experimental group accordingly (45 cases in each group). The control group intravenously received 20 mL/kg tranexamic acid before the incision was closed. The experimental group was given 1 g of tranexamic acid orally before anesthesia, 6 h and 12 h after the operation. Results. The experimental group witnessed better perioperative indexes in relation to the control group. The experimental group displayed better postoperative coagulation function indexes as compared to the control group (P<0.05). Remarkably lower postoperative vascular endothelial function indexes in the experimental group than in the control group were observed. The experimental group experienced a markedly lower incidence of deep vein thromboembolism in comparison with the control group (P<0.05). The postoperative knee society score (KSS) score of the experimental group was significantly higher than that of the control group. A significantly higher postoperative modified rivermead mobility index (MRMI) score was yielded in the experimental group in contrast to the control group (P<0.05). The experimental group obtained lower numerical rating scale (NRS) scores at T2 and T3 as compared to the control group. Conclusion. Oral tranexamic acid is a suitable alternative for patients undergoing TKA in terms of reducing the blood transfusion rate, relieving pain, and accelerating the recovery of the patient’s limbs