Biology subjects

Parissi, V.

Publications and source records attributed to Parissi, V..

3 recordsLinked to original sources

bioRxiv2021

In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human ACE2 complex, viral entry and cell fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a novel computational model of the SARS-CoV-2 S/ACE2 complex as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context, and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.

microbiology↗

bioRxiv2020

Analysis of the intrinsic chromatin binding property of HIV-1 integrase and its regulation by LEDGF/p75 using human chromosomes spreads

Retroviral integration requires the stable insertion of the viral genome into the host chromosomes. During this process, the functional integration complex must associate with cellular chromatin via the interaction between retroviral integrase and nucleosomes. The final association between the HIV-1 integration complex and the nucleosomal target DNA remains unclear and may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To date, there is no experimental system allowing the direct monitoring of this protein association with chromatin to depict the molecular mechanism of this process fully. To investigate this and understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase further, we used both biochemical approaches and an unedited chromosome-binding assays. Our study revealed that retroviral IN has an intrinsic ability to bind and recognize specific chromatin regions even in the absence of its cofactor. We also showed that this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromatin regions. Using these approaches, we also better determined the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. In addition to supporting a chromatin-binding function of the integrase protein acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate, our work precisely elucidates the mechanism of action of LEDGF/p75 in this crucial integration step.

microbiology↗

bioRxiv2020

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Integrons are genetic systems conferring to bacteria a rapid adaptation capability. The integron integrase is able to capture, stockpile and shuffle novel functions embedded in cassettes. This involves the recognition of both substrates, the attI site, and the cassette associated attC sites. Integrons can be sedentary and chromosomally located (SCI) or, carried by conjugative plasmids (Mobile Integron, MI), hence favoring their dissemination among bacteria. Here, for the first time, we investigate the cassette recruitment in the Vibrio cholerae SCI during conjugation and natural transformation. We demonstrated that horizontally transferred cassette can be recruited inside the chromosomal integron. The endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of single-stranded cassettes during conjugation and natural transformation, to mediate significant cassette insertion. We demonstrate that the attIA insertion is preferential, despite the presence of 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to adaptive selection. We also show that the RecA protein is critical for cassette recruitment in V. cholerae SCI but not in MIs. Moreover, a contrario to MIs, the V. cholerae SCI is not active in others bacterial hosts. MIs might have evolved from the SCIs by overcoming host factors, which would explain their large dissemination in bacteria and their role in the antibioresistance expansion.

molecular biology↗