Innate imune response against retrovirus

Lucia González: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Natalia Ibañez: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Marcelo Mateus: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Karina Romero: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Otto Pritsch: Docente supervisor. Departamento de Inmunobiología, Facultad de Medicina, Universidad de la República, Uruguay. Contacto: Otto Pritsch. E-mail: [email protected] retrovirus son un diverso grupo de virus que se encuentran en los vertebrados. Su importancia biomédica radica en que son capaces de infectar humanos, produciendo importantes problemas de salud. El virus de la inmunodeficiencia humana (VIH) es capaz de producir un estado de inmunodeficiencia en el huésped determinando el desarrollo de enfermedades oportunistas en estadio avanzados de la enfermedad. Frente a la entrada de un retrovirus al organismo, nuestro sistema inmune presenta como primera línea de defensa a la inmunidad innata. El resultado de esta respuesta es la inducción de interferones de tipo I (IFN tipo I) quienes generan un estado antiviral en la célula. Recientemente se ha ampliado la investigación sobre diferentes factores de restricción del huésped que forman parte de la inmunidad innata antiviral determinando la inhibición de la replicación de los retrovirus. En esta revisión abordaremos las distintas vías de señalización implicadas en la función de estos facto res. Dentro de ellos, se mencionarán; el SAMHD1 que determina un agotamiento del pool celular de dNTP inhibiendo los pasos tempranos de la retrotranscripción en células infectadas; TREX1 que es considerado un factor de restricción del huésped antagónico ya que la ausencia del mismo resulta en la activación de una respuesta de interferón; APOBEC3 que media la restricción viral principalmente por un mecanismo de edición del DNA; TRIM5α que puede formar una estructura hexagonal por encima de la cápside, lo cual desestabilizaría el core viral; Tetherin que es capaz de bloquear la liberación de viriones de VIH.Retrovirus are a diverse group of viruses that can be founded in vertebrates. Its biomedical signifi cance is that they are able to infect humans, causing significant health problems. The human immuno deficiency virus (HIV) generates progressive failure of the immune system and allows opportunistic diseases to settle in advanced stages of the infection. The innate immune response is the first line of defense against the entrance of retrovirus. The result of this response is the induction of type I interfe ron which establish an antiviral state in the host cell. Recently, research has been widened to different host restriction factors that are part of the innate immune antiviral response which are able to suppress retrovirus replication. In this review we discuss different signaling pathways involved in the function of these factors. We mention, the SAMHD1which determines a cell depletion dNTP pool inhibiting early steps of reverse transcription in cells infected by retrovirus; TREX1 which is considered a res triction factor of the antagonistic host as the absence thereof results in the activation of an interferon response; APOBEC3 that mediates viral restriction mainly by an edition of DNA mechanism; TRIM5α which can form an hexagonal structure above the capsid, which would destabilize the viral core; and finally Tetherin which is able to block the liberation of HIV virions

Serine/threonine protein kinase PrkA of the human pathogen Listeriamonocytogenes: Biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thrphosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His6-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.Agencia Nacional de Investigación e Innovació

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species

Drug repurposing screening validated by experimental assays identifies two clinical drugs targeting SARS-CoV-2 main protease

The COVID-19 pandemic prompted several drug repositioning initiatives with the aim to rapidly deliver pharmacological candidates able to reduce SARSCoV- 2 dissemination and mortality. A major issue shared by many of the in silico studies addressing the discovery of compounds or drugs targeting SARS-CoV- 2 molecules is that they lacked experimental validation of the results. Here we present a computer-aided drug-repositioning campaign against the indispensable SARS-CoV-2 main protease (MPro or 3CLPro) that involved the development of ligand-based ensemble models and the experimental testing of a small subset of the identified hits. The search method explored random subspaces of molecular descriptors to obtain linear classifiers. The best models were then combined by selective ensemble learning to improve their predictive power. Both the individual models and the ensembles were validated by retrospective screening, and later used to screen the DrugBank, Drug Repurposing Hub and Sweetlead libraries for potential inhibitors of MPro. From the 4 in silico hits assayed, atpenin and tinostamustine inhibited MPro (IC50 1 μM and 4 μM, respectively) but not the papain-like protease of SARSCoV- 2 (drugs tested at 25 μM). Preliminary kinetic characterization suggests that tinostamustine and atpenin inhibit MPro by an irreversible and acompetitive mechanisms, respectively. Both drugs failed to inhibit the proliferation of SARSCoV- 2 in VERO cells. The virtual screening method reported here may be a powerful tool to further extent the identification of novel MPro inhibitors. Furthermore, the confirmed MPro hits may be subjected to optimization or retrospective search strategies to improve their molecular target and anti-viral potency.Laboratorio de Investigación y Desarrollo de Bioactivo

Expression, purification, and characterization of bovine leukemia virus-like particles produced in Drosophila S2 cells

Material complementario: https://www.frontiersin.org/articles/10.3389/fviro.2021.756559/full#supplementary-materialBovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects cattle worldwide. In Uruguay, it is estimated that more than 70% of dairy cattle are infected, causing serious economic losses due to decreased milk production, increased calving interval, and livestock losses due to lymphosarcoma. Several attempts to develop vaccine candidates that activate protective immune responses against BLV were performed, but up to date, there is no vaccine that ensures efficient protection and/or decreased viral transmission. The development and application of new vaccines that effectively control BLV infection represent amajor challenge for countries with a high prevalence of infection. In this study, we generated two Drosophila melanogaster S2 stable cell lines capable of producing BLV virus-like particles (BLV-VLPs). One of them, BLV-VLP1, expressed both Gag and Env wild-type (Envwt) full-length proteins, whereas BLV-VLP2 contain Gag together with a mutant form of Env non-susceptible to proteolytic maturation by cellular furin type enzymes (EnvFm).We showed that Envwt is properly cleaved by cellular furin, whereas EnvFm is produced as a full-length gp72 precursor, which undergoes some partial cleavage. We observed that said mutation does not drastically affect its expression or its entry into the secretory pathway of S2 insect cells. In addition, it is expressed on the membrane and retains significant structural motifs when expressed in S2 insect cells. Morphology and size of purified BLV-VLPs were analyzed by transmission electron microscopy and dynamic light scattering, showing numerous non-aggregated and approximately spherical particles of variable diameter (70–200 nm) as previously reported for retroviral VLPs produced using different expression systems. Furthermore, we identified two N-glycosylation patterns rich in mannose in EnvFm protein displayed on VLP2. Our results suggest that the VLPs produced in Drosophila S2 cells could be a potential immunogen to be used in the development of BLV vaccines that might contribute, in conjunction with other control strategies, to reduce the transmission of the virus.CSIC I+D 2014ANII: ALI_1_2016_2_129851; POS_NAC_2015_1_109471PEDECIBA-FOCEM: COF 03/11CAP: BFPD_2020_1#2814383

Kinetics of bovine leukemia virus aspartic protease reveals its dimerization and conformational change

The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional −1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Fo¨ rster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.CSIC: I+D 201

What have we learned from a case of convalescent plasma treatment in a two-time kidney transplant recipient COVID-19 patient? A case report from the perspective of viral load evolution and immune response

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, can have a wide range of clinical manifestations, ranging from asymptomatic disease to potentially life-threatening complications. Convalescent plasma therapy has been proposed as an effective alternative for the treatment of severe cases. The aim of this study was to follow a two-time renal transplant patient with severe COVID-19 treated with convalescent plasma over time from an immunologic and virologic perspective. A 42-year-old female patient, who was a two-time kidney transplant recipient, was hospitalized with COVID-19. Due to worsening respiratory symptoms, she was admitted to the intensive care unit, where she received two doses of convalescent plasma. We analyzed the dynamics of viral load in nasopharyngeal swab, saliva, and tracheal aspirate samples, before and after convalescent plasma transfusion. The levels of pro-inflammatory cytokines and antibody titers were also measured in serum samples. A significant decrease in viral load was observed after treatment in the saliva and nasopharyngeal swab samples, and a slight decrease was observed in tracheal aspirate samples. In addition, we found evidence of an increase in antibody titers after transfusion, accompanied by a decrease in the levels of several cytokines responsible for cytokine storm.Fondo para la Convergencia Estructural del MercosurAmbassade de France à MontevideoAgencia Nacional de Investigación e InnovaciónCentro Latinoamericano de BiotecnologíaInstitut Pasteur de Montevide

What have we learned from a case of convalescent plasma treatment in a two-time kidney transplant recipient COVID-19 patient? A case report from the perspective of viral load evolution and immune response

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, can have a wide range of clinical manifestations, ranging from asymptomatic disease to potentially life-threatening complications. Convalescent plasma therapy has been proposed as an effective alternative for the treatment of severe cases. The aim of this study was to follow a two-time renal transplant patient with severe COVID-19 treated with convalescent plasma over time from an immunologic and virologic perspective. A 42-year-old female patient, who was a two-time kidney transplant recipient, was hospitalized with COVID-19. Due to worsening respiratory symptoms, she was admitted to the intensive care unit, where she received two doses of convalescent plasma. We analyzed the dynamics of viral load in nasopharyngeal swab, saliva, and tracheal aspirate samples, before and after convalescent plasma transfusion. The levels of pro-inflammatory cytokines and antibody titers were also measured in serum samples. A significant decrease in viral load was observed after treatment in the saliva and nasopharyngeal swab samples, and a slight decrease was observed in tracheal aspirate samples. In addition, we found evidence of an increase in antibody titers after transfusion, accompanied by a decrease in the levels of several cytokines responsible for cytokine storm