DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to acute respiratory distress syndrome, and death in the most severe cases. Immune dysregulation with altered innate cytokine responses is thought to contribute to disease severity. Here, we characterized in depth host cell responses against SARS-CoV-2 in primary human airway epithelia (HAE) and immortalized cell lines. Our results demonstrate that primary HAE and model cells elicit a robust induction of type I and III interferons (IFNs). Importantly, we show for the first time that melanoma differentiation associated gene (MDA)-5 is the main sensor of SARS-CoV-2 in lung cells. IFN exposure strongly inhibited viral replication and de novo production of infectious virions. However, despite high levels of IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication in lung cells, contrary to what was previously reported in intestinal epithelial cells. Altogether, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.IMPORTANCE Mammalian cells express sensors able to detect specific features of pathogens and induce the interferon response, which is one of the first line of defenses against viruses and help controlling viral replication. The mechanisms and impact of SARS-CoV-2 sensing in lung epithelial cells remained to be deciphered. In this study, we report that despite a high production of type I and III interferons specifically induced by MDA-5-mediated sensing of SARS-CoV-2, primary and immortalized lung epithelial cells are unable to control viral replication. However, exogenous interferons potently inhibited replication, if provided early upon viral exposure. A better understanding of the ambiguous interplay between the interferon response and SARS-CoV-2 replication is essential to guide future therapeutical interventions.

Reactivation of latent HIV-1 in vitro using an ethanolic extract from Euphorbia umbellata (Euphorbiaceae) latex.

Euphorbia umbellata (E. umbellata) belongs to Euphorbiaceae family, popularly known as Janauba, and its latex contains a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present in E. umbellata latex alcoholic extract that are able to increase HIV transcription and reactivate virus from latency models. This activity is probably mediated by NF-kB activation followed by nuclear translocation and binding to the HIV LTR promoter. In addition, E. umbellata latex extract induced the production of pro inflammatory cytokines in vitro in human PBMC cultures. This latex extract also activates latent virus in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+ T lymphocytes. Together, these results indicate that the phorbol esters present in E. umbellata latex are promising candidate compounds for future clinical trials for shock and kill therapies to promote HIV cure and eradication.

Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions.

Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.

2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1.

SAMHD1 restricts HIV-1 replication in myeloid and quiescent CD4(+) T cells. Here, we show that SAMHD1 restriction activity is regulated by phosphorylation. SAMHD1 interacts with cyclin A2/cdk1 only in cycling cells. Cyclin A2/CDK1 phosphorylates SAMHD1 at the Threonine 592 residue both in vitro and in vivo. Phosphorylation of SAMHD1 Thr592 correlates with loss of its ability to restrict HIV-1. Indeed, while PMA treatment of proliferating THP1 cells results in reduced Thr592 phosphorylation, activation of resting peripheral blood mononuclear cells (PBMCs) and purified quiescent CD4(+) T cells results in increased phosphorylation of SAMHD1 Thr592. Interestingly, we found that treatment of cells by type 1 interferon reduced Thr592 phosphorylation, reinforcing the link between the phosphorylation of SAMHD1 and its antiviral activity. Unlike wild-type SAMHD1, a phosphorylation-defective mutant was able to restrict HIV-1 replication in both PMA-treated and untreated cells. Our results uncover the phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 as a key regulatory mechanism of its antiviral activity.

Genotypic analysis of the gp41 HR1 region from HIV-1 isolates from enfuvirtide-treated and untreated patients.

OBJECTIVE: To evaluate the polymorphisms and resistance mutations in gp41 HR1 region of HIV-1. METHODS: The study included 28 HIV-positive patients undergoing enfuvirtide (ENF) treatment or not from Porto Alegre, Rio Grande do Sul state, and Rio de Janeiro, Rio de Janeiro state, between 2006 and 2009. Resistance mutations and polymorphisms of the gp41 HR1 region were detected using the genomic DNA of 12 ENF-untreated patients and 16 patients in ENF treatment, encompassing subtypes B, C, and F1. Sample subtypes were determined by neighbor-joining phylogenetic analysis with a Kimura's two-parameter correction. RESULTS: A high prevalence of polymorphisms unrelated to resistance was observed. Among ENF-untreated patients, 16% showed mutations related with resistance. Among patients in ENF treatment, 50% had resistance-related mutations. Overall, 17% of all isolates showed the N42S polymorphism related to ENF hypersusceptibility. The presence of ENF resistance mutations in the group of treated patients reduced viral load. The V38A substitution was the most frequent among treatment-experienced patients followed by the G36D/E, N42D, and V38M substitutions. CONCLUSIONS: The V38A substitution in the gp41 HR region was the most common resistance mutation among ENF-treated patients and was associated with increased viral load.

Construção e caracterização de vírus febre amarela (FA) 17D recombinantes expressando o domínio III da proteína e de dengue 2 na região intergênica e NS1 do vírus FA 17D / Construction and characterization of virus yellow fever (FAN) 17D recombinant expressing domain III of the protein and of dengue 2 in intergenic region and NS1 of the virus FAN 17D

A dengue é uma arbovirose transmitida ao homem por meio da picada de insetos vetores que albergam o vírus dengue (gênero Flavivirus, família Flaviviridae). Este vírus apresenta-se como 4 sorotipos (den-1 a den-4), antigenicamente distintos, embora todos possuam mesma epidemiologia e causem os mesmos sintomas. A infecção por dengue é caracterizada por amplo espectro de manifestações clínicas e sua re-emergência é responsável por grande impacto na saúde pública mundial. Apesar de diversas tentativas para o desenvolvimento de uma vacina contra dengue, nenhuma ainda está disponível. Neste trabalho utilizamos o vírus vacinal da Febre Amarela 17D (FA 17D) para expressão do domínio III (DIII) da proteína de envelope (E) de den-2, na região intergênica E/NS1, baseado nas boas propriedades da vacina contra FA. A escolha do DIII den-2 justifica-se por ele estar associado a processos de infecção celular e por ser o principal indutor de resposta imune mediada por anticorpos neutralizantes dirigida à partícula viral. O estudo de alinhamento do DIII de diferentes genótipos e asua modelagem molecular permitiram uma melhor caracterização desta região em relação à sua variabilidade, mutantes de escape de neutralização e sítios de ligação a receptores celulares. A abordagem de expressão do DIII da proteína E de den-2 no genoma do vírus FA 17D baseou-se na montagem do cassete de expressão contendo motivos duplicados de aminoácidos conservados, flanqueadores da região intergênica E e NSI (Bonaldo et al., 2007), permitindo desta forma, o correto processamento da poliproteína viral em associação ao retículo endoplasmático (RE) celular. Foram gerados dois diferentes vírus FA recombinantes que albergavam a inserção heteróloga correspondente ao DIII den-2, indicando que esta inserção não acarretou em um grande comprometimento da estrutura e função virais. Estes vírus FA recombinantes diferiam entre si quanto à porção C-terminal do cassete de expressão, onde um deles...Flavivírus.