Biology subjects

Tramontano, E.

Publications and source records attributed to Tramontano, E..

6 recordsLinked to original sources

bioRxiv2024

Screening Envelope Genes Across Primate Genomes Reveals Evolution and Diversity Patterns of Endogenous Retroviruses

Endogenous Retroviruses (ERVs) are integrated into the host DNA as result of ancient germ line infections, majorly by now extinct exogenous retroviruses. Accordingly, vertebrates genomes contain thousands of ERV copies, providing "fossil" records for the ancestral retroviral diversity and its evolution within the host. Like exogenous retroviruses, ERV proviral sequence consists of gag, pro-pol, and env genes flanked by long terminal repeats (LTRs). In particular, the characterization of env gene changes over time allows both to understand ERVs evolutionary trajectory and to infer their potential role in host physiology, including their pathological involvement. Despite their potential impact on the host, a detailed characterization has been performed for few domesticated env genes, while a comprehensive survey of their abundance and diversity among primates genomes is still lacking. The study primarily focused on ERVs env that are known to be present in hominoid species, given their well-characterized nature and availability in public databases. Hence using these env sequences, we reconstructed 32 Env sequences representing the prototypes of these ancestral proteins in Class I, Class II, and Class III HERVs. These reconstructed Envs were then used for similarity search, phylogenetic analysis, and examination of recombination events occurred within primates genomes that were applied to 55 primate species across the Catarrhini and Platyrrhini parvorders. Through such comprehensive pipeline, we reconstituted a phylogenetic distribution of ERV based specifically on the env genes, showing that the ERVs have been prevalent and widely distributed across the primate lineage. We observed for the first time the presence of the HML (human mouse mammary tumor virus-like) group in the Platyrrhini parvorder, indicating initiation of spread of HML supergroup before the split between New World Monkeys (NWM) and Old World Monkeys (OWM) i.e. even before 40 mya. Importantly, we confirmed interclass and intra-class env recombination events confirming the phenomenon of "env snatching" among primates ERVs. As a result, we demonstrate that tracing the diversity patterns of ERVs env provides relevant insights into the retroviral evolutionary history of ERVs in Catarrhini and Platyrrhini parvorders. Overall, our findings reveal that env recombination contributes to the diversification of ERVs, thereby broadening our comprehension of retroviral and primate evolution.

microbiology↗

bioRxiv2023

Resolving the pharmacological redox-sensitivity of SARS-CoV-2 PLpro in drug repurposing screening enabled identification of the competitive GRL-0617 binding site inhibitor CPI-169

The SARS CoV-2 Papain-Like protease has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its capacity to antagonize host immune response. Targeting PLpro function is recognized as a promising mechanism to modulate viral replication whilst supporting host immune responses. However, development of PLpro specific inhibitors remains challenging. Upcoming studies revealed the limitation of reported inhibitors by profiling them through a pipeline of enzymatic, binding and cellular activity assays showing unspecific activity. GRL-0617 remained the only validated molecule with demonstrated anti-viral activity in cells. In this study we refer to the pitfalls of redox-sensitivity of PLpro. Using a screening-based approach to identify inhibitors of PLpro proteolytic activity, we made extensive efforts to validate the active compounds over a range of conditions and readouts, emphasising the need for comprehensive orthogonal data when profiling putative PLpro inhibitors. The remaining active compound CPI-169, showed to compete with GRL-0617 in NMR-based experiments, suggesting to share a similar binding mode, opening novel design opportunities for further developments as antiviral agents. Author summaryThe increasing knowledge about SARS-CoV-2 allowed the development of multiple strategies to contain the spread of COVID-19 infection. Nevertheless, effective antiviral pharmacological treatments are still rare and viral evolution allowed a fast adaptation and escape from available containment methods. The papain like protease (PLpro) has now become the next most promising SARS-CoV-2 therapeutic due to its multiple functions in virus replication cycle and antagonization of host immune response. However, due to inherent flexibility and sensitivity of this enzyme specific inhibitors are rare. Here we report on a screening strategy using repurposing of known drugs that takes into account PLpro characteristics to identify new inhibitors, showing the success of the approach by identifying CPI-169 that competitive targets the well described GRL-0617 inhibitor binding pocket of PLpro and helping to design further antiviral agents.

molecular biology↗

bioRxiv2022

Nirmatrelvir treatment blunts the development of antiviral adaptive immune responses in SARS-CoV-2 infected mice

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir - an orally available inhibitor of the 3-chymotrypsin-like cysteine protease - has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using a mouse model of SARS-CoV-2 infection, we show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

immunology↗

bioRxiv2020

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 M and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 [A], showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/422677v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17ca2aeorg.highwire.dtl.DTLVardef@19c5159org.highwire.dtl.DTLVardef@1a0adf6org.highwire.dtl.DTLVardef@1fd05cd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure.C_FLOATNO Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray. C_FIG

molecular biology↗

bioRxiv2019

RNA-seq transcriptome analysis reveals LTR-retrotransposons modulation in Human Peripheral Blood Mononuclear Cells (PBMCs) after in vivo Lipopolysaccharides (LPS) injection

Human Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) are retroviral sequences that integrated into the germline cells millions year ago. Transcripts of these LTR-retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. In this work, we focused on the HERVs/MaLRs expression and modulation in a scenario of immune system activation. We used a public dataset of Human Peripheral Blood Mononuclear Cells (PBMCs) RNA-seq from 15 healthy participants to a clinical trial before and after the exposure to Lipopolysaccharide (LPS), for which we established an RNA-seq workflow for the identification of expressed and modulated cellular genes and LTR-retrotransposon elements. ImportanceWe described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4 % of the LTR-retrotransposons loci were expressed, and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERVs and MaLRs loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci differentially expressed checking their genomic context of insertion and, interestingly, we found a general co-localization with genes that are involved and modulated in the immune response, as consequence of LPS stimulation. The analyses of HERVs and MaLRs expression and modulation show that this LTR-retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR-retrotransposons and the immune host response.

microbiology↗

bioRxiv2019

Mutation of Ebola virus VP35 Ser129 uncouples interferon antagonist and replication functions

Ebolaviruses are non-segmented, negative-sense RNA viruses (NNSVs) within the order Mononegavirales that possess the multifunctional virion protein 35 (VP35), a major determinant of virulence and pathogenesis that is indispensable for viral replication and host innate immune evasion. VP35 is functionally equivalent to the phosphoprotein (P) of other mononegaviruses such as rhabdoviruses and paramyxoviruses. Phosphorylation of the P protein is universally regarded as functionally important however, a regulatory role(s) of phosphorylation on VP35 function remains unexplored. Here, we identified a highly conserved Ser129 residue near the homo-oligomerization coiled coil motif, which is essential for VP35 functions. Affinity-purification MS followed by post-translational modification (PTM) analysis predicted phosphorylation of Ser129. Co-immunoprecipitation, cross-linking, and biochemical characterization studies revealed a moderately decreased capacity of VP35-S129A to oligomerize. Functional analysis showed that Ser-to-Ala substitution of Ebola virus (EBOV) VP35 did not affect IFN inhibitory activity but nearly abolished EBOV minigenome activity. Further coimmunoprecipitation studies demonstrated a lost interaction between VP35-S129A and the amino terminus of the viral polymerase but not between viral nucleoprotein (NP) or VP35-WT. Taken together, our findings provide evidence that phosphorylation modulates VP35 function, supporting VP35 as a NNSV P protein and providing a potentially valuable therapeutic target.\n\nImportanceEbola virus (EBOV) can cause severe disease in humans. The 2013-2016 West African epidemic and the two recent outbreaks in the Democratic Republic of the Congo underscore the urgent need for effective countermeasures, which remain lacking. A better understanding of EBOV biology and the modulation of multifunctional viral proteins is desperately needed to develop improved therapeutics. We provide evidence here that function of virion protein 35 (VP35) is modulated by phosphorylation of Ser129, a conserved residue among other ebolavirus species. These findings shed light on EBOV biology and present a potential target for broad acting anti-ebolavirus therapeutics.

microbiology↗