Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19). It emerged from China in December 2019 and rapidly spread across the globe, causing a pandemic with unprecedented impacts on public health and economy. Therefore, there is an urgent need for the development of curative treatments and vaccines. In humans, COVID-19 pathogenesis shows a wide range of symptoms, from asymptomatic to severe pneumonia. Identifying animal models of SARS-CoV-2 infection that reflect the clinical symptoms of COVID-19 is of critical importance. Nonhuman primates (NHPss) correspond to relevant models to assess vaccine and antiviral effectiveness. This review discusses the use of NHPs as models for COVID-19 research, with focus on the pathogenesis of SARS-CoV-2 infection, drug discovery and pre-clinical evaluation of vaccine candidates.

A cure for chronic hepatitis B requires eliminating or permanently silencing covalently closed circular DNA (cccDNA). A pivotal target of this approach is the hepatitis B virus (HBV) X protein (HBx), which is a key factor that promotes transcription from cccDNA. However, the HBx structure remains unsolved. Here, we present the cryoelectron microscopy structure of HBx in complex with DDB1, which is an essential complex for cccDNA transcription. In this structure, hydrophobic interactions within HBx were identified, and mutational analysis highlighted their importance in the HBV life cycle. Our biochemical analysis revealed that the HBx–DDB1 complex directly interacts simultaneously with NSE3, which is a component of the SMC5/6 complex, and Spindlin1. Additionally, HBx–DDB1 complex dynamics were explored via high-speed atomic force microscopy. These findings provide comprehensive insights into the structure and function of HBx in HBV replication.

Background: Chronic hepatitis B virus (HBV) infection remains a major global health burden due to the persistence of covalently closed circular DNA (cccDNA), a stable episomal viral reservoir resistant to current antiviral therapies. The transcriptional activity of cccDNA depends on its chromatin organization, yet the contribution of histone variants to this regulation remains poorly understood. Methods: Using mass spectrometry–based proteomics on native cccDNA purified from infected primary human hepatocytes, we identified the histone variant H2A.Z and its chaperone SRCAP as cccDNA-associated proteins. Functional analyses combining shRNA-mediated silencing, chromatin immunoprecipitation (ChIP), and ATAC-seq were performed in HBV-infected HepG2-NTCP cells and primary human hepatocytes to characterize their roles in cccDNA formation and transcription. Results: Depletion of H2A.Z.1, H2A.Z.2, or SRCAP reduced both HBV RNA levels and cccDNA formation. H2A.Z recruitment to cccDNA correlated with the establishment of active chromatin marks (H3K4me3) and enhanced RNA polymerase II loading, promoting an open and transcriptionally active chromatin state. In addition, we identified BRD2, an H2A.Z-associated transcriptional co-activator, as a positive regulator of HBV transcription. shRNA mediated depletion and pharmacological degradation of BRD2 using the PROTAC ARV-771 reduced HBV RNA levels, supporting its potential as an antiviral target. Conclusion: Our findings uncover a critical role of the H2A.Z variant and SRCAP complex in cccDNA formation and transcriptional activation. By facilitating chromatin accessibility and RNA polymerase II recruitment, H2A.Z establishes an epigenetic environment favorable to HBV persistence. Targeting H2A.Z-associated co-activators such as BRD2 may represent a promising strategy to silence cccDNA transcription and achieve functional HBV cure.

Chronic hepatitis B virus infection persists as a global health crisis, driving life-threatening liver pathologies such as hepatocellular carcinoma. Central to HBV’s resilience is the covalently closed circular DNA (cccDNA), a viral minichromosome that orchestrates viral transcription and sustains infection. Dissecting cccDNA-driven transcription remains a formidable challenge due to the dense overlap of viral open reading frames, which generates RNA transcripts with shared sequences, complicating precise quantification and hindering efforts to unravel HBV’s transcriptional regulation. To address this bottleneck, we developed a multiplexed assay named ddPCR for Overlapping Transcripts (ddOTs) capable of simultaneously quantifying all major HBV RNA species, including splice variants, with unprecedented specificity and sensitivity. This method overcomes current limitations by leveraging the high-resolution power of ddPCR to deconvolute overlapping transcripts, enabling direct interrogation of individual promoter/enhancer activities and RNA stability. By offering a cost-effective, scalable solution for precise RNA profiling in rare biological samples, this breakthrough tool unlocks new avenues for exploring cccDNA biology and accelerating antiviral drug development.