Biomedical subjects

Alessia Ruggieri

Publications and source records attributed to Alessia Ruggieri.

5 recordsLinked to original sources

PubMed2026

ModiCal: A Targeted Calibration Workflow for Site-Specific m5C Validation by Nanopore Direct RNA Sequencing.

Accurate identification of RNA 5-methylcytidine (m5C) at the single-nucleotide resolution remains a central challenge in nanopore direct RNA sequencing (DRS). Current global scanning and modification-aware basecalling methods enable transcriptome-wide profiling but often yield high false-positive rates and lack site-specific accuracy. To address this, we repurposed ModiDeC, originally a de novo multimodification classifier, into a targeted, high-precision validation tool for RNA modification sites with prior biochemical knowledge. This was implemented through a three-step calibration workflow that alternates between biochemical and computational modules using the well-characterized m5C2278 site in 25S rRNA as a starting point. Baseline training uses short synthetic RNAs carrying either a methylated or unmodified C2278 as ground truth, followed by IVT-derived calibration and validation in methyltransferase knockout yeast. The baseline model accurately detected the bona fide m5C2278 site but initially produced off-target predictions. Iterative retraining with unmodified IVT signals progressively reduced and ultimately eliminated false positives while maintaining a strong signal at the bona fide site. The final model retained enzyme-dependent detection in wild-type versus knockout yeast and, when explicitly targeted, was also able to detect the second rRNA site, C2870, which remained invisible in the initial analysis. Application to native human prerRNA processing intermediates further resolved two distinct m5C deposition regimes on 28S rRNA, while generalization to dengue virus genomic RNA confirmed that the same calibration logic transfers across diverse RNA contexts. Together, this study establishes a reproducible and transferable framework that integrates biochemical validation with iterative neural network refinement, providing a route toward reliable site-specific m5C confirmation by nanopore direct RNA sequencing.

RNA Methylation↗

PubMed2004

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians.

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.

Amino Acid Sequence↗

PubMed2003

SIV Vectors.

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Animals↗

PubMed2002

The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors.

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.-L. Blond, D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel-Fernandez, B. Mandrand, F. Mallet, and F.-L. Cosset, J. Virol. 74:3321-3329, 2000) and that it can pseudotype human immunodeficiency virus cores (D. S. An, Y. Xie, and I. S. Y. Chen, J. Virol. 75:3488-3489, 2001). By using cell-cell fusion and pseudotype virion infection assays, we found that HERV-W Env efficiently uses both hASCT2 and the related transporter hASCT1 (gene name, SLC1A4) as receptors. In addition, although HERV-W Env mediates only slight syncytium formation or infection of mouse cells, it utilizes the mouse transporters mASCT1 and mASCT2 when their sites for N-linked glycosylation are eliminated by mutagenesis. Consistent with their role as a battlefield in host-virus coevolution, the viral recognition regions in ASCT1 and ASCT2 of humans and mice are highly divergent compared with other regions of these proteins, and their ratios of nonsynonymous to synonymous nucleotide sequence changes are extremely large. The recognition of ASCT1 and ASCT2 despite this divergence of their sequences strongly suggests that the use of both receptors has been highly advantageous for survival and evolution of the HERV-W family of retroviruses.

Amino Acid Sequence↗

PubMed2002

Relationship between SU subdomains that regulate the receptor-mediated transition from the native (fusion-inhibited) to the fusion-active conformation of the murine leukemia virus glycoprotein.

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.

Binding Sites↗