Circular RNAs (circRNAs) are single-stranded RNAs that form a covalently closed loop without 5' and 3' ends. From their own genomes, RNA viruses can encode circRNAs that play important roles during infection, but whether this is the case for HIV remains unclear. Here we detect an HIV-1-encoded viral circRNA (termed circHIV) in plasma from 18 people living with HIV and in infected primary cells and T-cell lines using RT-qPCR and northern blot. We find that this viral circRNA is packaged within HIV-1 virions and binds to the HIV-1 Tat protein upon expression in cells, which enhances transcription from the viral promoter, as shown by RNA immunoprecipitation and in vitro pulldown assays. Our study sheds light on a previously overlooked component of HIV-1 transcription, revealing the ability of circRNAs to act as transcriptional regulators that drive viral pathogenesis.

Plasmids are mobile genetic elements that can rapidly spread across bacterial populations, promoting the dissemination of antimicrobial resistance (AMR) genes in bacteria. They are enriched in insertion sequences (IS), which are small transposable elements able to translocate between genetic locations. Here we combined experimental, bioinformatic and computational approaches to investigate the role of plasmids promoting AMR through IS-mediated gene inactivation. We find that plasmid pOXA-48, which encodes two IS1 elements, increases the rate of resistance acquisition to multiple antibiotics in clinical strains of Klebsiella pneumoniae through IS1-mediated gene disruption. Screening of genome databases confirmed that the inactivation of genes through plasmid-encoded IS elements is an extended mechanism of AMR evolution. Both our experiments and computational model revealed that conjugative plasmids can promote this route of AMR acquisition while invading complex bacterial communities. Overall, we show that conjugative plasmids contribute to AMR not only through the dissemination of resistance genes, but also through IS-mediated gene inactivation.

The emergence of SARS-CoV-2 Omicron variants has led to viral escape from many clinically approved monoclonal antibodies (mAbs) due to rapid evolution of the receptor-binding domain (RBD). Co-circulation of SARS-CoV-2 variants with unique sets of antigenic substitutions has further complicated therapeutic mAb discovery. New approaches are needed to rapidly discover and characterize mAbs with preferred specificity and functional characteristics. Here we describe and perform epitope-focused mAb discovery using glycan-masked antigens. We isolated and expressed a panel of 303 mAbs, some of which potently neutralize divergent Omicron subvariants by targeting the class 3 antigenic site on SARS-CoV-2 RBD. Epitope mapping of these antibodies revealed a spectrum of cross-reactivity and differential recognition of the class 3 site, validating the utility of this enrichment approach for targeted mAb discovery. Together, this work rationally designs glycan-masked engineered RBDs and uses them to isolate mAbs that potently neutralize antigenically divergent SARS-CoV-2 variants.

Yellow fever virus (YFV) continues to threaten human and wildlife populations in the Americas, yet its transmission at the forest-urban interface remains unclear. Here we integrate ground- and canopy-level mosquito surveillance, systematic monitoring of non-human primate carcasses and viral metagenomics to describe the dynamics of a sylvatic YFV outbreak in a 186-hectare Atlantic Forest fragment embedded within metropolitan São Paulo, Brazil, between 2017 and 2018. Our analyses reveal that transmission was primarily driven by a single genetic cluster introduced during a period of high abundance of the main vector, Haemagogus leucocelaenus mosquitoes. A near-complete hepatitis A virus genome was detected in a YFV-infected howler monkey, suggesting potential co-infections at the human-wildlife interface. Phylogenetic and epidemiological modelling estimated a basic reproduction number, R0, for sylvatic yellow fever of 8.2 (95% CI 5.1-12.2), substantially higher than previous estimates for urban outbreaks. Our findings demonstrate that multisource surveillance could provide actionable early warnings in regions at risk for zoonotic spillover.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems present a barrier to prophage acquisition by restricting invading phages or by inducing autoimmune cleavage of integrated prophage DNA. The RNA-sensing type VI CRISPR nuclease Cas13 mediates non-specific RNA cleavage upon recognition of phage lytic transcripts, but how this system influences the temperate phage life cycle remains unknown. Here we report that the Listeria seeligeri type VI-A CRISPR system restricts the lytic cycle of temperate phages but tolerates prophage acquisition and interferes with prophage induction through a non-abortive mechanism. During attempts at induction, Cas13 activation forces prophage re-integration, thus maintaining lysogeny. We also find that during polylysogenic induction, Cas13 acts specifically, restricting only the targeted phage, in contrast to its behaviour during lytic replication. Our findings show that Cas13 elicits a unique response to each stage of the temperate phage life cycle, enabling type VI CRISPR hosts to acquire potentially beneficial prophages while mitigating lysis.

Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.

Targeting the gut microbiota is a promising strategy to enhance the efficiency of cancer immunotherapy; however, success has been limited. Here we combined metagenomic analysis and in silico prediction to identify bacterial species associated with immunotherapy response in patients with non-small-cell lung cancer. We constructed a defined consortium (RCom) of 15 bacterial species, most of which were isolated from responder patient faeces, associated with improved clinical response to anti-programmed cell death protein 1 (PD-1) treatment. Metabolic models and in vitro experiments revealed that RCom is a stable and cooperative community, and in vivo experiments showed that RCom engrafts and produces immunomodulatory metabolites. Oral administration of RCom improved the anti-tumour activity of anti-PD-1 by increasing the intratumoural infiltration and cytotoxic function of CD8+ T cells in syngeneic tumour models and across mice with heterogeneity in baseline gut microbiota composition. RCom supplementation also limited anti-PD-1 resistance in mice conferred by faecal microbiota transplantation from individual non-responsive patients. These findings suggest that RCom is a potential adjuvant to improve responsiveness to anti-PD-1 therapy in cancer.

Trichoderma fungi support sustainable agriculture by suppressing plant diseases and improving crop performance. However, emerging pathogenicity of Trichoderma warrants further ecological and genetic characterization. Here we used machine learning to correlate genomic data from 37 Trichoderma strains with over 140 phenotypic traits, spanning metabolic versatility, biotic interactions, stress tolerance and reproductive strategies. We determined Trichoderma to be an ancient, genetically cohesive and physiologically diverse genus with spores capable of germination in water and dispersal via air and water droplets. Metabolic preferences indicate universal adaptation to mycoparasitism and to niches like arboreal microbial mats, alongside broader saprotrophic versatility. Our analyses are consistent with character displacement among close relatives and convergent evolution in distant lineages, with both processes shaping ecological plasticity and traits including dispersal modes, terrestrialization or endophytism. Our findings reveal that while some Trichoderma species show traits of biosafety concern, its vast ecophysiological diversity enables the development of safe, targeted bioeffectors.