Biology subjects

Scudero, O. B.

Publications and source records attributed to Scudero, O. B..

4 recordsLinked to original sources

bioRxiv2026

Virus-Driven Proximity Proteomics Identifies p400 as a Restriction Factor Poised on Incoming Viral Genomes

Immediate-early viral proteins must rapidly counteract host defenses directed at incoming viral genomes, yet the protein interaction networks that mediate this remain unresolved. This gap persists largely because immediate-early proteins are expressed at low abundance during this stage of infection, making them challenging to investigate with standard approaches. Here, we present a virus-driven proximity proteomics framework to investigate immediate-early phase virus-host interactions in an authentic infection context. An isogenic P2A control virus untethers a miniTurbo biotin ligase from the viral protein under investigation, effectively modeling bait abundance and background changes from a matched infection context. Continuous biotin labeling from the earliest hours of infection further amplifies detection of transient, low-abundance interactions characteristic of this phase. Using the adenovirus E1A hub protein as a benchmark, we recovered the majority of known E1A interactors and identified over 150 high-confidence interactions. Applying this strategy to the immediate-early phase, we resolved the E1A interactome and identified the p400 chromatin remodeling complex as its dominant target. We show p400 associates with incoming viral genomes, represses gene expression, and regulates persistent infection. These data identify p400 as a poised host restriction factor and establish a temporally resolved proximity proteomics strategy transferable to other immediate-early viral proteins.

microbiology↗

bioRxiv2026

Differential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutants

Recognition of dsRNA triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus infection differentially modulates dsRNA sensors and RNP granule assembly. Infection with splicing-defective {Delta}E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While {Delta}VA-induced granules were PKR-dependent, {Delta}E4 mutants induced RLB-like granules independently of PKR and RNase L. In these cells, granule assembly coincided with translational arrest independent of eIF2 phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.

cell biology↗

bioRxiv2023

The Respiratory Syncytial Virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly

The M2-2 protein from the respiratory syncytial virus (RSV) is a 10 kDa protein expressed by the second ORF of the viral gene M2. During infection, M2-2 has been described as the polymerase cofactor responsible for promoting genome replication. This function was first inferred by infection with a mutant virus lacking the M2-2 ORF, in which viral genome presented delayed accumulation in comparison to wild-type virus. In accordance with this phenotype, it has been recently shown that M2-2 promotes changes in interactions between the polymerase and other viral proteins at early stages of infection. Despite its well-explored role in the regulation of the polymerase activity, little has been made to investigate the relationship of M2-2 with cellular proteins. In fact, a previous report showed poor recruitment of M2-2 to viral structures, with the protein being mainly localized to the nucleus and cytoplasmic granules. To unravel which other functions M2-2 exerts during infection, we expressed the protein in HEK293T cells and performed proteomic analysis of co-immunoprecipitated partners, identifying enrichment of proteins involved with regulation of translation, protein folding and mRNA splicing. In approaches based on these data, we found that M2-2 expression downregulates eiF2 phosphorylation and inhibits stress granules assembly under arsenite induction. In addition, we also verified that M2-2 inhibits translation initiation, and is targeted for proteasome degradation, being localized to granules composed by defective ribosomal products at the cytoplasm. These results suggest that besides its functions in the regulation of genome replication, M2-2 may exert additional functions to contribute to successful RSV infection. Author summaryExploring how viruses take control of the cellular machinery is a common strategy to understand the infection process and to identify targets for inhibition of virus replication. In this work we investigated the cellular functions of the protein M2-2 from the respiratory syncytial virus. Although this virus is an important pathogen responsible for respiratory infections in immunocompromised individuals, currently there are no vaccines or effective treatments to inhibit its infection. Our findings showed that the protein M2-2 interferes with protein synthesis, being able to downregulate the assembly of stress granules during stress stimuli. Besides, we verified the relationship between M2-2 and the proteasome machinery, which is responsible for protein degradation and is also involved with protein synthesis. These results present new functions for the protein M2-2, indicating additional mechanisms utilized by the virus to facilitate infection, providing new perspectives for the search of antiviral targets.

microbiology↗

bioRxiv2021

Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and {gamma}H2AX (another DNA damage marker) expression. We also observed an increase of PCNA and {gamma}H2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

molecular biology↗