Biology subjects

Petit, C. M.

Publications and source records attributed to Petit, C. M..

5 recordsLinked to original sources

bioRxiv2025

Subcellular determinants of orthoflavivirus protease activity

Orthoflaviviruses are small, enveloped, positive-sense RNA viruses that cause over 500 million infections globally each year for which there are no antiviral treatments. The viral protease is an attractive target for therapeutics due to its critical functions throughout infection. Many studies have reported on the structure, function, and importance of orthoflavivirus proteases; However, the molecular determinants for cleavage of intracellular substrates by orthoflavivirus proteases and how these factors affect viral fitness are unknown. In this study, we used our fluorescent, protease-activity reporter system to investigate the subcellular determinants involved in orthoflavivirus protease cleavage. By modifying our reporter platform, we identified endoplasmic reticulum (ER) subdomain localization and membrane proximity of the substrate cut site as two previously uncharacterized molecular determinants for cleavage. We also altered the amino acid composition of the reporter cut site to introduce sequences present at the cytoplasmic junctions within orthoflavivirus polyproteins and found that each protease processed the sequence located at the junction between NS4A and the 2K peptide least efficiently. Live-cell imaging revealed that cleavage of the NS4A|2K sequence is significantly delayed compared to the capsid cleavage sequence. We further determined that introducing a more efficient cleavage sequence into the NS4A|2K junctions of orthoflavivirus infectious clones abolished virus recovery. Overall, this study identifies ER subdomain localization and membrane proximity of the cut site as molecular determinants for cleavage by orthoflavivirus proteases and provides insight into the role that sequence specificity plays in the coordinated processing of the viral polyprotein and establishing productive infections. ImportanceOrthoflaviviruses are the most prevalent and dangerous arthropod-borne viruses (arboviruses) leading to over 500 million global infections annually. Orthoflavivirus infection can cause severe pathologies, including hemorrhagic conditions and neurological disease, that lead to hundreds of thousands of deaths each year. The viral protease complex, responsible for processing the viral polyprotein into its functional subunits, is an attractive target for antiviral therapeutic development. Despite extensive research efforts on these viral protein complexes, all protease inhibitor candidates have fallen short of clinical efficacy, highlighting a considerable gap in knowledge of the viral proteases complex intracellular activity. The significance of our research is in characterizing the subcellular determinants associated with orthoflavivirus protease cleavage efficiency and how these factors can influence viral fitness. These findings contribute to closing this gap in knowledge of the mechanisms of orthoflavivirus proteases which can ultimately lead to the successful development of targeted antivirals.

microbiology↗

bioRxiv2023

14-3-3 phosphorylation inhibits 14-3-3theta's ability to regulate LRRK2 kinase activity

LRRK2 mutations are among the most common genetic causes for Parkinsons disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3{theta} isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3{theta} phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3{theta} mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3{theta} mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3{theta} mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3{theta} phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3{theta} and phosphorylated T2524 LRRK2 was important for 14-3-3{theta}s ability to regulate kinase activity, as wildtype and S232A 14-3-3{theta} failed to reduce the kinase activity of G2019S/T2524A LRRK2. Molecular modeling showed that 14-3-3{theta} phosphorylation causes a partial rearrangement of its canonical binding pocket, thus affecting the interaction between 14-3-3{theta} and the C-terminus of LRRK2. We conclude that 14-3-3{theta} phosphorylation destabilizes the interaction of 14-3-3{theta} with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity.

neuroscience↗

bioRxiv2021

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs1-4, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits5. Here we show that water-soluble derivatives of -tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D--tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and {beta}-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of -tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp6. In summary, we conclude that solubilizing modifications to -tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection7-9.

microbiology↗

bioRxiv2020

Structure of nonstructural protein 1 from SARS-CoV-2.

The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1s importance in the CoV lifecycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral lifecycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 - 127) at 1.77[A] resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 - 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral lifecycle. IMPORTANCEThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus lifecycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77[A] resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

microbiology↗

bioRxiv2020

Influenza-induced oxidative stress sensitizes lung cells to bacterial toxin-mediated necroptosis

RationalePneumonia caused by Influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity. Previously we have shown that pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. ObjectivesDetermine the impact of IAV infection on bacterial PFT-mediated lung epithelial cell (LEC) necroptosis. Determine the molecular basis for increased sensitivity and if inhibition of necroptosis or oxidative stress blocks IAV sensitization of LEC to PFT. MethodsMice and cells were challenged with IAV followed by Spn. Necroptosis was monitored by measuring cell death at fixed time points post-infection and immunofluorescent detection of necroptosis. Wildtype mice and LEC were treated with necroptosis inhibitors. Necroptosis effector molecule MLKL deficiency was tested for infection synergy. Oxidative damage to DNA and lipids as result of infection was measured in vitro and in vivo. Necroptosis and anti-oxidant therapy efficacy to reduce disease severity was tested in vivo. Measurements and Main ResultsIAV synergistically sensitized LEC for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacological induction of oxidative stress sans virus sensitized cells for PFT-mediated necroptosis. Necroptosis inhibition reduced disease severity during secondary bacterial infection. ConclusionsIAV-induced oxidative stress sensitizes LEC for PFT-mediated necroptosis. This is a new molecular explanation for severe influenza-associated bacterial infections. Necroptosis inhibitors are potential therapeutic strategies to reduce IAV-primed bacterial pneumonia severity. SummaryHere we demonstrate that Influenza A virus (IAV) infection synergistically sensitizes lung cells to bacterial pore-forming toxin (PFT)-mediated necroptosis. Moreover, this contributes to the severity of lung injury that is observed during co- and secondary infection with Streptococcus pneumoniae. IAV-induced oxidative stress was identified as a key factor contributing to cell sensitization and induction of oxidative stress sans virus was sufficient to synergistically enhance susceptibility to PFT-mediated killing. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identifies necroptosis inhibition and antioxidant therapy as potential intervention strategies.

microbiology↗