Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

Traceability of SARS-CoV-2 transmission through quasispecies analysis.

During COVID-19 pandemic, consensus genomic sequences were used for rapidly monitor the spread of the virus worldwide. However, less attention was paid to intrahost genetic diversity. In fact, in the infected host, SARS-CoV-2 consists in an ensemble of replicating and closely related viral variants so-called quasispecies. Here we show that intrahost single nucleotide variants (iSNVs) represent a target for contact tracing analysis. Our data indicate that in the acute phase of infection, in highly likely transmission links, the number of viral particles transmitted from one host to another (bottleneck size) is large enough to propagate iSNVs among individuals. Furthermore, we demonstrate that, during SARS-CoV-2 outbreaks when the consensus sequences are identical, it is possible to reconstruct the transmission chains by genomic investigations of iSNVs. Specifically, we found that it is possible to identify transmission chains by limiting the analysis of iSNVs to only three well-conserved genes, namely nsp2, ORF3, and ORF7.

Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing.

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.

A Boron Delivery Antibody (BDA) with Boronated Specific Residues: New Perspectives in Boron Neutron Capture Therapy from an In Silico Investigation.

Boron Neutron Capture Therapy (BNCT) is a tumor cell-selective radiotherapy based on a nuclear reaction that occurs when the isotope boron-10 (10B) is radiated by low-energy thermal neutrons or epithermal neutrons, triggering a nuclear fission response and enabling a selective administration of irradiation to cells. Hence, we need to create novel delivery agents containing 10B with high tumor selectivity, but also exhibiting low intrinsic toxicity, fast clearance from normal tissue and blood, and no pharmaceutical effects. In the past, boronated monoclonal antibodies have been proposed using large boron-containing molecules or dendrimers, but with no investigations in relation to maintaining antibody specificity and structural and functional features. This work aims at improving the potential of monoclonal antibodies applied to BNCT therapy, identifying in silico the best native residues suitable to be substituted with a boronated one, carefully evaluating the effect of boronation on the 3D structure of the monoclonal antibody and on its binding affinity. A boronated monoclonal antibody was thus generated for specific 10B delivery. In this context, we have developed a case study of Boron Delivery Antibody Identification Pipeline, which has been tested on cetuximab. Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor used in the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer.