Jianli Tao

O’nyong-nyong virus (ONNV) is a mosquito-borne alphavirus responsible for large-scale epidemics in sub-Saharan Africa. As the closest evolutionary relative of Chikungunya virus (CHIKV), ONNV shares substantial genetic similarity and overlapping clinical manifestations with CHIKV. Mechanistic understanding of ONNV infection has therefore largely been extrapolated from CHIKV rather than directly established. However, ONNV exhibits distinct biological features, including predominant transmission by Anopheles mosquitoes and a clinical presentation characterized by prominent lymphadenopathy with limited acute joint edema. These distinctions underscore the need for an integrated synthesis of experimentally validated determinants of ONNV infection. In this review, we summarize current evidence on molecular and immunological factors regulating ONNV infection in mammalian hosts and mosquito vectors. We first discuss species-specific viral clearance, host dependency factors, intrinsic antiviral restriction mechanisms, protective innate immunity, inflammatory pathology, and mechanism-informed therapeutic strategies in mammalian hosts. We then examine stage-specific immune regulation in Anopheles mosquitoes, emphasizing mechanisms that constrain viral replication while permitting persistent infection and transmission. Finally, we discuss nsP3-dependent vector specificity and the potential contribution of alternative mosquito species to ONNV ecology. Together, this review provides an integrated framework for understanding how host factors, immune responses, and vector-specific adaptations shape ONNV infection, pathogenesis, and transmission.

Background: Achieving FAIR-compliant computational research in bioinformatics is systematically undermined by two compounding challenges that existing tools leave unresolved: long-term reproducibility and accessibility. Standard package managers re-download dependencies from live repositories at every build, making environments vulnerable to library disappearance and version drift, and pinning a package version does not pin the versions of its transitive dependencies, causing divergences between builds performed at different points in time. Compounding this, packages from repositories such as CRAN, Bioconductor, and PyPI frequently omit critical system-level dependencies from their installation metadata, leaving users to manually discover which underlying library is missing or which version is required. Beyond these technical failures, constructing a truly reproducible environment demands expertise in containerization making reproducibility in practice a privilege and not a standard. Findings: We present REBEL (Reproducible Environment Builder for Explicit Library Resolution), a framework that addresses both challenges through three dependency inference heuristics: (i) Deep Inspection of source code, (ii) Fuzzy Matching against a manually curated knowledge base, and (iii) Conservative Dependency Locking. The resolved dependency stack is then archived into a self-contained local store, enabling offline and deterministic rebuilds at any future time. We compared the installation of 1,000 randomly sampled CRAN packages in isolated Docker containers versus the standard package manager and REBEL resolved 149 of 328 standard installation failures (45.4%). Moreover through its DockerBuilder component, REBEL further generates fully reproducible Docker images from a plain text requirements file, making deterministic environment construction accessible without expertise in containerization. Conclusions: REBEL provides a practical foundation for FAIR-compliant, long-term reproducible bioinformatics analyses, making deterministic environment construction accessible to researchers regardless of their technical background. REBEL is freely available at https://github.com/Rebel-Project-Core

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and B-cell receptor (BCR) pathway inhibitors such as idelalisib and ibrutinib are currently established therapies for CLL. Although effective, these drugs frequently lead to resistance, but the mechanisms are still not fully understood. Activation-induced cytidine deaminase (AID) is a B-cell enzyme essential for antibody diversification. However, it can also introduce off-target mutations, leading to genomic instability. This study investigates whether treatment with BCR pathway inhibitors increases AID activity in CLL and whether this activity contributes to the development of drug resistance. Peripheral blood samples from CLL patients were collected before and after treatment with idelalisib or ibrutinib. Targeted sequencing was used to identify mutations in known AID off-target genes. Concurrently, AID-wild type (AID-WT) and AID-knockout (AID-KO) CLL cell lines were established and subsequently exposed to escalating doses of BCR pathway inhibitors to develop drug-resistant models. In patient samples, treatment with BCR pathway inhibitors was associated with an increase in AID-dependent mutations in off-target genes, including BCL2, MYC, and IRF8. The in vitro models efficiently recapitulated the patients’ data, as only AID-WT CLL cells accumulated mutations in the same AID off-target genes after drug exposure. However, no mutations were detected in genes that could mediate drug resistance. We conclude that BCR pathway inhibitors enhance AID mutational activity in CLL, but this does not appear to be directly involved in driving drug resistance. AID-targeted loci may nonetheless serve as biomarkers for monitoring genomic instability during treatment and inform further study.

The enhancer of zeste homolog 2 (EZH2) histone methyltransferase inhibitors tazemetostat and valemetostat recently have received approval for clinical use in follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. In follicular lymphoma, the expression of activation-induced cytidine deaminase (AID) is responsible for increased mutational signatures and genomic instability. Because EZH2 inhibitors induce epigenetic and transcriptional changes in normal and lymphoma B cells, we studied whether these inhibitors could alter the pattern of AID-mediated chromosomal translocations. In this study, we showed that treatment with EZH2 inhibitors did not significantly change AID expression or AID-dependent chromosomal translocation frequency when used as monotherapy in either CH12F3 mouse B cells or MEC-1 human B cells. In contrast, when combined with phosphoinositide 3-kinase δ (PI3Kδ) inhibition, which enhances AID expression, EZH2 inhibition significantly increased the frequency of chromosomal translocations when compared with either EZH2 or PI3Kδ inhibition alone both in mouse CH12F3 cells and human MEC-1 cells. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were DNA ligase IV (Ligase4) deficient. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3 trimethylation at lysine 27 (H3K27me3) while concurrently enhancing the active histone modification H3 acetylation at lysine 27 (H3K27ac), thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. These findings highlight the impact of drugs that induce epigenetic changes to influence chromosomal translocations, and demonstrate the genetic safety of EZH2 inhibitors as monotherapy while highlighting the increased risk of genomic instability when used in cells prone to translocations, such as B cells with high AID levels or DNA repair deficiency.

Mosquito-borne diseases are a group of illnesses caused by pathogens transmitted by mosquitoes, and they are globally prevalent, particularly in tropical and subtropical regions. Pathogen transmission occurs during mosquito blood feeding, a process in which mosquito saliva plays a crucial role. Mosquito saliva contains a variety of biologically active proteins that facilitate blood feeding by preventing blood clotting, promoting vasodilation, and modulating the host’s immune and inflammatory responses. These effects create an environment conducive to pathogen invasion and dissemination. Specific mosquito salivary proteins (MSPs) can promote pathogen transmission through mechanisms that either regulate hosts’ anti-infective immune responses or directly enhance pathogens’ activity. Strategies targeting these MSPs have emerged as an innovative and promising approach for the control of mosquito-borne diseases. Meanwhile, the diversity of these proteins and their complex interactions with the host immune system necessitate further research to develop safer and more effective interventions. This review examines the functional diversity of MSPs and their roles in disease transmission, discusses the advantages and challenges of strategies targeting these proteins, and explores potential future directions for research in this area.

CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.

CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

Mitochondrial antiviral-signaling protein (MAVS) transmits signals from RIG-I-like receptors after RNA virus infections. However, the mechanism by which MAVS activates downstream components, such as TBK1 and IKKα/β, is unclear, although previous work suggests the involvement of NEMO or TBK1-binding proteins TANK, NAP1, and SINTBAD. Here, we report that MAVS-mediated innate immune activation is dependent on TRAFs, partially on NEMO, but not on TBK1-binding proteins. MAVS recruited TBK1/IKKε by TRAFs that were pre-associated with TBK1/IKKε via direct interaction between the coiled-coil domain of TRAFs and the SDD domain of TBK1/IKKε. TRAF2^−/−3^−/−5^−/−6^−/− cells completely lost RNA virus responses. TRAFs’ E3 ligase activity was required for NEMO activation by synthesizing ubiquitin chains that bound to NEMO for NF-κB and TBK1/IKKε activation. NEMO-activated IKKα/β were important for TBK1/IKKε activation through IKKα/β-mediated TBK1/IKKε phosphorylation. Moreover, individual TRAFs differently mediated TBK1/IKKε activation and thus fine-tuned antiviral immunity under physiological conditions. Innate immunity is the first line of defense against virus infection. RIG-I-like receptors (RLRs) recognize various viral RNA from RNA viruses and initiate host antiviral responses to produce type I interferons (IFNs) and other cytokines. RLRs sense distinct types of viruses by sharing a common adaptor protein called mitochondrial antiviral-signaling protein (MAVS). Although it has been well studied how RLRs recruit and activate MAVS upon virus infection, it remains to be elucidated how MAVS activates its downstream components, including kinases TBK1/IKKε and the IKK complex. Here, by using TANK^−/−NAP1^−/−SINTBAD^−/−, TRAF2^−/−3^−/−5^−/−6^−/− and TRAF2^−/−3^−/−5^−/−6^−/−NEMO^−/− 293T cells combined with reconstitution experiments, we discovered that MAVS recruited TBK1/IKKε via TRAFs through pre-associated TRAFs-TBK1/IKKε complex. TBK1/IKKε activation required both TRAFs-mediated TBK1 autophosphorylation and TRAFs-NEMO-IKKβ-mediated TBK1 phosphorylation. We demonstrated that TRAFs’ E3 ligase activity was solely required for NEMO and IKKα/β activation. IKKα/β were crucial for both TBK1 and NF-κB activation. Our results thus demonstrated that MAVS activates TBK1/IKKε through TRAFs in both NEMO-dependent and independent manner. Importantly, a minimal amount of IFNs was produced independent of NF-κB activation during virus infection and that individual TRAFs differently mediated TBK1/IKKε activation, thus fine-tuned antiviral immunity under physiological conditions.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediates innate immune responses against invading pathogens, or against self-dsDNA, which causes autoimmune disorders. Upon nonspecific binding of cytosolic B–form DNA, cGAS synthesizes the second messenger 2′3′-cGAMP and triggers STING-dependent signaling to produce type I IFNs. The cGAS comprises less-conserved N-terminal residues and highly conserved nucleotidyltransferase/Mab21 domains. The function and structure of the well-conserved domains have been extensively studied, whereas the physiological function of the N-terminal domain of cGAS is largely uncharacterized. In this study we used a single-molecule technique combined with traditional biochemical and cellular assays to demonstrate that binding of nonspecific dsDNA by the N-terminal domain of cGAS promotes its activation. We have observed that the N terminus of human cGAS (hcGAS-N160) undergoes secondary structural change upon dsDNA binding in solution. Furthermore, we showed that the hcGAS-N160 helps full length hcGAS to expand the binding range on λDNA and facilitates its binding efficiency to dsDNA compared with hcGAS without the 160 N-terminal residues (hcGAS-d160). More importantly, hcGAS-N160 endows full length hcGAS relatively higher enzyme activity and stronger activation of STING/IRF3-mediated cytosolic DNA signaling. These findings strongly indicate that the N-terminal domain of cGAS plays an important role in enhancing its function.