Biology subjects

Reguera, J.

Publications and source records attributed to Reguera, J..

5 recordsLinked to original sources

bioRxiv2026

Stepwise assembly and mechanical stability of nsP3 Helical Scaffolds in Chikungunya Virus

Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus that replicates its genome through a viral replication complex (RC) essential for successful infection. The RC consists of four non-structural proteins: nsP1, nsP2, nsP3, and nsP4. In this study, we focused on nsP3, which comprises three distinct domains: the alphavirus unique domain (AUD), the long and intrinsically disordered hypervariable domain (HVD), and the enzymatic macrodomain (MD). Notably, nsP3 assembles into helicoidal macrostructures, termed helical scaffolds (HSs), which are components of the CHIKV replication complex and are involved in the formation of macromolecular traps in the cytoplasm of the infected cell called Alphagranules (AGs). HSs form tubular helical scaffolds assembled through the AUD, with the MD and HVD flexibly deployed to the scaffold exterior. Despite their functional importance, the mechanisms governing HS formation remain poorly understood. Using High-Speed Atomic Force Microscopy (HS-AFM) and Electron Microscopy (EM) we achieved new findings on the structure, mechanics and assembly of these macrostructures. Our results show the nsP3 monomers follow intermediate steps before completing the assembly into full helical scaffolds, with each nsP3 domain contributing differently to this process. The macrodomain (MD) provides mechanical stability, yet it fractures under tens of piconewtons of force. This rigidity acts as a mechanical regulator that constrains the position of the HVD, which in turn mediates interactions with host factors and neighboring HSs. Through this interplay, nsP3 achieves both structural cohesion and functional adaptability, promoting the higher-order organization of HSs into honeycomb-like networks.

molecular biology↗

bioRxiv2026

Hantaan virus polymerase has an in vitro Terminal Nucleotidyl Transferase activity that is conserved across the Bunyaviricetes class

Bunyaviricetes is a class of segmented negative strand RNA viruses (sNSV) that includes causative agents of severe zoonotic diseases resulting in hemorrhagic fever in humans and livestock. Hantaan virus (HTNV), family Hantaviridae, is an example of life-threatening bunyavirus transmitted by rodents which leads to Hantavirus hemorrhagic fever with renal syndrome in humans. Infection relies on the replication and transcription of their tripartite genomes through multifunctional RNA-dependent RNA polymerases (RdRp) in the context of ribonucleoproteins (RNPs) made of one viral RNA fragment, one RdRp and multiple copies of viral nucleoproteins. The RdRp, also known as L protein, performs replication by de novo initiation through a prime-and-realign mechanism while transcription initiates through a cap snatching mechanism, that engages a cap-binding domain and an endonuclease (EN) domain present in the L protein C and N terminal regions, respectively. In this work we show that HTNV L protein has a terminal nucleotidyl transferase (TNTase) activity over double-stranded RNA substrates (dsRNA) in vitro. We show that the TNTase activity is also present in other bunyaviral L proteins by comparing HTNV L protein activity with La Crosse (LACV, family Orthobunyaviridae) and Crimean Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) L proteins. Our results show that TNTase activity appears to be a general feature of Bunyaviruses and report the differences on substrate and nucleotide specificity for each family of virus.

biochemistry↗

bioRxiv2022

Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). 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 SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering 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 intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals. Significance statementHere we present biochemical and structural characterization of the capping pathway carried out by the Chikungunya virus nsP1 capping pores. We provide five Cryo-EM structures representative of the different steps of the reaction. These structures reveal the molecular determinants and dynamics associated with the alphavirus capping process. In addition, we biochemically show the RNA capping specificity and the reversibility of the reaction which allow nsP1 to cap and decap RNAs and to release intermediates of the reaction. These data provide a new biochemical clues on the enzymatic activity of nsP1 capping pores and a new structural landscape that will be instrumental for the design of effective antivirals targeting the viral RNA capping for blocking the infection.

molecular biology↗

bioRxiv2022

Structural basis of Ty1 integrase tethering on RNA polymerase III for targeted DNA integration

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.

molecular biology↗

bioRxiv2020

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational changes inherent to its function. We uncover the coordinated movement of the polymerase priming loop, lid domain and C-terminal region required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. The revealed structural details and dynamics of functional elements will be instrumental for structure-based development of compounds that inhibit RNA synthesis by the polymerase.

biophysics↗