Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N ⁷ methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m ⁷ GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m ⁷ GTP in the active site, and the subsequent covalent transfer of m ⁷ GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile.

Here we report the characterization of 17T2, a SARS-CoV-2 pan-neutralizing human monoclonal antibody isolated from a COVID-19 convalescent individual infected during the first pandemic wave. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA⁺ memory B cell, with a broad neutralizing activity against former and new SARS-CoV-2 variants, including XBB.1.16 and BA.2.86 Omicron subvariants. Consistently, 17T2 demonstrates in vivo prophylactic and therapeutic activity against Omicron BA.1.1 infection in K18-hACE2 mice. Cryo-electron microscopy reconstruction shows that 17T2 binds the BA.1 spike with the RBD in “up” position and blocks the receptor binding motif, as other structurally similar antibodies do, including S2E12. Yet, unlike S2E12, 17T2 retains its neutralizing activity against all variants tested, probably due to a larger RBD contact area. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 as a potential candidate for future clinical interventions.

Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication. Structural determination of RNPs can provide crucial information on the molecular mechanisms of these processes, paving the way for the development of new, more effective strategies to control and prevent the spread of ssRNAv diseases. In this scenario, cryogenic electron microscopy (cryoEM), relying on the technical and methodological revolution it has undergone in recent years, can provide invaluable help in elucidating how these macromolecular complexes are organized, packaged within the virion, or the functional implications of these structures. In this review, we summarize some of the most prominent achievements by cryoEM in the study of RNP and nucleocapsid structures in lipid-enveloped ssRNAv.