Baculovirus vectors induce the production of interferons in swine: Their potential in the development of antiviral strategies

The huge variety of viruses affecting swine represents a global threat. Since vaccines against highly contagious viruses last several days to induce protective immune responses, antiviral strategies for rapid control of outbreak situations are needed. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an insect virus, has been demonstrated to be an effective vaccine vector for mammals. Besides the ability to display or transduce heterologous antigens, it also induces strong innate immune responses and provides IFN-mediated protection against lethal challenges with viruses like foot-and-mouth disease virus (FMDV) in mice. Thus, the aim of this study was to evaluate the ability of AcMNPV to induce IFN production and elicit antiviral activity in porcine peripheral blood mononuclear cells (PBMCs). Our results demonstrated that AcMNPV induced an IFN-α-mediated antiviral activity in PBMCs in vitro. Moreover, the inoculation of AcMNPV in piglets led to the production of type I and II IFNs in sera from inoculated animals and antiviral activities against vesicular stomatitis virus (VSV) and FMDV measured by in vitro assays. Finally, it was demonstrated that the pseudotyping of AcMNPV with VSV-G protein, but not the enrichment of the AcMNPV genome with specific immunostimulatory CpG motifs for the porcine TLR9, improved the ability to induce IFN-α production in PBMCs in vitro. Together, these results suggest that AcMNPV is a promising tool for the induction of IFNs in antiviral strategies, with the potential to be biotechnologically improved.Fil: Molina, Guido Nicolás. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Otero, Ignacio. Universidad Maimónides; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Molinari, Maria Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors. IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.Fil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Molina, Guido Nicolás. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; ArgentinaFil: López, María Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Prenatal Hyperandrogenization Induces Metabolic and Endocrine Alterations Which Depend on the Levels of Testosterone Exposure

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life

Role of the cytoplasmic nucleic acids of the baculovirus AcMNPV in the production of a IFN-I in non-immune mammalian cells

El baculovirus AcMNPV es un virus envuelto, con genoma DNA de doble cadena, patógeno de insectos. Posee un complejo ciclo de replicación bifásico en el que participan dos fenotipos: los virus brotados (BV) y los virus derivados de los cuerpos de oclusión (ODV), los cuales son estructural y funcionalmente distintos. Los BV son capaces de transducir genes bajo la regulación de un promotor adecuado en células de mamífero, aunque no pueden replicar su genoma en este hospedador. Dentro de las aplicaciones de los baculovirus como herramienta biotecnológica en mamíferos, se destacan su uso para el desarrollo de vacunas, para el delivery de genes y como inmunomoduladores. Se ha descripto una potente respuesta inmune innata inducida por BV en mamíferos, capaz de montar un estado antiviral inespecífico por vías independientes de TLR. Así, este trabajo de Tesis tiene como finalidad estudiar la participación de los ácidos nucleicos baculovirales citosólicos en la producción de un estado antiviral en células no inmunes de mamífero. En primer lugar, mediante ensayos de transcripción con baculovirus recombinantes se demostró que el citoplasma es el principal destino que alcanza el genoma baculoviral en distintos tipos celulares no inmunes. A continuación, se observó que los BV -pero no los ODV- son capaces de producir un estado antiviral y un aumento de IFN β en fibroblastos murinos. Además, se determinó que baculovirus pseudotipados con la proteína G (VSV) ingresan a la célula de manera más eficiente pero no producen mayores niveles de IFN β. Finalmente, se estudió la participación de distintos sensores citosólicos de DNA en células murinas y en células humanas infectadas con BV y se observó que la enzima RNA Pol III no está relacionada en el establecimiento del estado antiviral. La vía cGAS-STING en células murinas fue estudiada mediante edición génica por CRISPR-Cas9, mientras que en células epiteliales humanas se estudió mediante transcomplementación de cGAS o cGAS y STING en las células HEK293 y HEK293 T, respectivamente. Los resultados mostraron que STING es una proteína necesaria para el establecimiento de un estado antiviral en células de mamífero. Además, al menos dos vías de señalización distintas impactan en STING y contribuyen al estado antiviral por baculovirus. La detección del genoma viral por el sensor cGAS induce la respuesta celular más potente y es necesaria para la producción de IFN β. Además, la activación de STING, de un modo cGAS independiente, da lugar a un estado antiviral en el cual, en células epiteliales humanas, participa la producción de IFN λ1. En conclusión, los resultados de esta Tesis muestran que el genoma del baculovirus AcMNPV disponible en alta proporciones en el citosol en el contexto de infección de células no inmunes de mamífero, desempeña un papel relevante en el establecimiento de un estado antiviral y en la producción de IFN I y III a través del impacto en la vía de reconocimiento de ácidos nucleicos cGAS-STING.The baculovirus AcMNPV is an enveloped virus with a double-stranded DNA genome and is a pathogen of insects. It possesses a complex biphasic replication cycle with two phenotypes involved: budded virus (BV) and occlusion-derived virus (ODV), which are structurally and functionally different. The BV is capable of transducing genes under the control of an adequate promoter in mammalian cells, although it cannot replicate its genome in this host. Among the applications of baculoviruses as a biotechnological tool in mammals, is worth mentioning their use for vaccine development, gene delivery and as immunomodulators. A strong innate immune response induced by BV in mammals has been described, and it is capable of inducing an unspecific antiviral state, independent of TLR pathways. Thus, this thesis aims to study the role of baculoviral cytoplasmic nucleic acids in the production of an antiviral state in non-immune mammalian cells. First, transcription assays with recombinant baculovirus showed that cytoplasm is the main destination reached by baculoviral genome in different non-immune cell lines. Next, it was observed that BVs -but not ODVs- are capable of producing an antiviral state and an increase of IFN β in murine fibroblasts. Furthermore, we determined that baculoviruses pseudotyped with protein G (VSV) enter the cell more efficiently without the production of higher levels of IFN β. Finally, we studied the involvement of different cytosolic DNA sensors in murine and human cells infected with BV. It was observed that RNA Pol III does not participate in the establishment of the antiviral state. We then studied the cGAS-STING pathway with CRISPR-Cas9 gene editing in murine cells, whereas in human epithelial cells was studied with trans-complementation of cGAS or cGAS and STING in HEK293 and HEK293 T, respectively. The results showed that STING is required for the establishment of an antiviral state in mammalian cells. Moreover, at least two different signaling pathways have an impact on STING and contribute to the baculovirus induced antiviral state. The detection of the viral genome by cGAS sensing induces the strongest cellular response and it is necessary for the production of IFN β. Additionally, the cGAS-independent STING activation produces an antiviral state in human epithelial cells where the production of IFN λ1 is involved. In conclusion, the results of this thesis show that the genome of the baculovirus AcMNPV available in high levels in the cytosol during the infection of nonimmune mammalian cells, has a relevant role in the establishment of an antiviral state and in the production of IFN I and III through its impact in the nucleic acids sensing pathway of cGAS-STING.Fil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Link between metformin and the peroxisome proliferator-activated receptor γ pathway in the uterine tissue of hyperandrogenized prepubertal mice

Chronic hyperandrogenism alters the peroxisome proliferator-activated receptor γ (PPARγ) pathway in the uterine tissue of prepubertal mice. The gene and protein expression of PPARγ is not modified, but the gene and protein expression of 12-lipoxygenase (12-LOX), an enzyme that synthesizes PPARγ ligands, is decreased. The antihyperglycemic drug metformin can prevent this adverse effect.Fil: Elia, Evelin Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Pustovrh, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Chile; ChileFil: Amalfi, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Devoto, Luigi. Universidad de Chile; ChileFil: Motta, Alicia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response

In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.Fil: Tavarone, Maria Eugenia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Molina, Guido Nicolás. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peralta, Andrea Verónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Molinari, Maria Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

P26 enhances baculovirus gene delivery by modulating the mammalian antiviral response

Poxins are poxviral proteins that act by degrading 2´3´-cGAMP, a key molecule of cGAS-STING axis that drives and amplifies the antiviral response. Previous works have described some poxin homologous among lepidopteran and baculoviral genes. In particular, P26, a poxin homologous from AcMNPV retains the 2´3´-cGAMP degradation activity in vitro. In this work, we demonstrated that the antiviral activity triggered by baculovirus was disrupted by the transient expression of P26 in murine and human cell lines, and the effect of this action is not only on IFN-β production but also on the induction of IFN-λ. Besides, we proved P26 functionality in a stable-transformed cell line where the protein was constitutively expressed, preventing the production of IFN-β induced by baculovirus and resulting in an improvement in the transduction efficiency by the attenuation of the antiviral activity. Finally, we incorporated P26 into budded virions by capsid display or passive incorporation, and the results showed that both strategies resulted in an improvement of 3–17 times in the efficiency of transgene expression in murine fibroblasts. Our results suggest that the incorporation of P26 to budded baculoviral vectors is a very promising tool to modulate negatively the innate antiviral cellular response and to improve the efficiency of gene delivery in mammalian cells.Instituto de BiotecnologíaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Amalfi, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Plastine, María Del Pilar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Plastine, María Del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Lopez, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gravisaco, María José. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Gravisaco, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Characterisation and transferability of transcriptomic microsatellite markers for Nothofagus species

Discriminant molecular markers are required for research on population genetics, as well as evolutionary studies involving identification of hybrids and parental species, or detection of the genome regions under selection. We provide a set of 27 transcriptomic microsatellite markers (SSRs) for South American Nothofagus species, derived from 73 Nothofagus alpina (=N. nervosa) annotated unigenes. Rates of cross-amplification ranged from 22% to 37%. Genetic characterisation of 22 transcriptomic SSRs for N. alpina and N. obliqua reveals low genetic variability, due to the general occurrence of one major allele at each locus, and high specificity, with few alleles shared between species (14%). At inter-species level 95% of loci were discriminant, with a total G''st over loci of 0.9, indicating that alleles were mostly fixed for all loci in both species. At intra-species level the number of markers with significant differentiation was 2.5 times higher for N. obliqua than for N. alpina populations. Moreover, transcriptomic SSRs showed higher performance compared with published anonymous microsatellites isolated from genome sequences without annotation. This set of transcriptomic microsatellites will be useful to the scientific community working on conservation and evolutionary aspects of Nothofagus species.Fil: El Mujtar, Verónica Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: López, María Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pomponio, María Florencia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torales, Susana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentin

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.Instituto de BiotecnologíaFil: Costa Navarro, Guadalupe Soledad. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Arneodo Larochette, Joel Demian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin