Biology subjects

Tumiotto, C.

Publications and source records attributed to Tumiotto, C..

4 recordsLinked to original sources

bioRxiv2025

Molecular basis for the regulation of retroviral nucleosomal integration by chromatin compaction

Cellular chromatin represents the first nonreversible contact point between the genomes of incoming infectious agents, such as integrative viruses, and their hosts. The integration of retroviral genomes requires a functional association between the viral integration complex (intasome) and host chromatin, mediated through multiple interfaces between the integrase, target DNA, and histone components of the nucleosome. Previous studies have shown that these associations are regulated by cellular factors, such as LEDGF/p75 for lentiviruses, and by the degree of compaction of the chromatin surrounding the insertion site. However, the molecular mechanisms underlying the regulation of access to local nucleosomal functional interfaces remain elusive. In this study, we dissected how incoming intasomes engage nucleosome surface to achieve efficient integration. Combining biochemical approaches with molecular docking analysis, we demonstrated that HIV-1 and PFV intasomes distinctly and specifically interact with nucleosome surfaces. Mapping these interfaces onto a compacted trinucleosomal structure and simulating the dynamic docking of the intasome at these sites revealed how neighboring nucleosomes modulate the functional binding of the HIV-1 intasome to the catalytic target nucleosome by masking these functional interfaces. In contrast, the lower susceptibility of the PFV intasome to chromatin compaction was due to the persisting accessibility of active nucleosomal interfaces. Together, these data provide the first molecular and structural insights into how chromatin compaction influence retroviral integration. Our results especially show how nucleosome-intasomes docking sites participate in modulating the sensitivity of the retroviral integration to chromatin structure. Overall, our data reveal that HIV-1 and PFV integration rely on nucleosomes with distinct structural and functional properties at the insertion site to form an active strand transfer complex. This work further demonstrated that retroviruses have evolved distinct strategies to engage suitable chromatin structures for efficient integration highlighting a divergence in retroviral adaptation mechanisms.

microbiology↗

bioRxiv2023

Reference materials for SARS-CoV-2 molecular diagnostic: validation of encapsulated synthetic RNAs for room temperature storage and shipping

The Coronavirus pandemic unveiled the unprecedented need for diagnostic tests to rapidly detect the presence of pathogens in the population. Real-time RT-PCR and other nucleic acid amplification techniques are accurate and sensitive molecular techniques that necessitate positive controls. To meet this need, Twist Bioscience has developed and released synthetic RNA controls. However, RNA is an inherently unstable molecule needing cold storage, costly shipping, and resource-intensive logistics. Imagene provides a solution to this problem by encapsulating dehydrated RNA inside metallic capsules filled with anhydrous argon, allowing room temperature and eco-friendly storage and shipping. Here, RNA controls produced by Twist were encapsulated (RNAshells) and distributed to several laboratories that used them for COVID-19 detection tests by amplification. One RT-LAMP procedure, four different RT-PCR devices and 6 different PCR kits were used. The amplification targets were genes E, N; RdRp, Sarbeco-E and Orf1a/b. RNA retrieval was satisfactory, and the detection was reproducible. RNA stability was checked by accelerated aging. The results for a 10-year equivalent storage time at 25 {degrees}C were not significantly different from those for unaged samples. This room temperature RNA stability allows the preparation and distribution of large strategic batches which can be stored for a long time and used for standardization processes between detection sites. Moreover, it makes it also possible to use these controls for single use and in the field where large temperature differences can occur. Consequently, this type of encapsulated RNA controls, processed at room temperature, can be used as reference materials for the SARS-Cov-2 virus as well as for other pathogens detection.

molecular biology↗

bioRxiv2022

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome

Retroviral integration into cell chromatin requires the formation of integrase-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes. To further study this mechanism we developed an alphaLISA approach using the prototype foamy virus (PFV) intasome and human nucleosome. This system allowed us to monitor the association between both partners and investigate the protein/protein and protein/DNA interactions engaged in the association with chromatin. Using this approach, we next screened the chemical OncoSET library and selected small molecules that could modulate the intasome/nucleosome complex. Molecules were selected as acting either on the DNA topology within the nucleosome or on the integrase/histone tail interactions. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, structural and cellular approaches. These drugs were shown to inhibit PFV and HIV-1 integration in vitro as well as HIV-1 infection in primary PBMCs cells. Our work provides new information about intasome-nucleosome interaction determinants and paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring.

microbiology↗

bioRxiv2020

HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen. METHODSSamples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis. RESULTSHIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were from 50 million to 350 million cells. CONCLUSIONClones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

genomics↗