Interação vírus-hospedeiro: a ativação de componentes da via de resposta a proteínas mal dobradas (UPR) pelos Vaccinia virus Guarani P1 (GP1V) e Passatempo (PSTV)

Abstract

The Poxviridae family has globally recognized members that infect a range of vertebrate and invertebrate hosts that includes humans. Poxviruses are classified into two subfamilies: the Entomopoxvirinae comprises poxviruses that infect insects, while the Choropoxvirinae includes the ones that infect vertebrates. The subfamily Chordopoxvirinae currently contains eighteen genera and the genus Orthopoxvirus is one of the most investigated. Since 1999, the Vaccinia virus (VACV) has been reported in outbreaks involving humans, cattle and horses, among other animals. Brazilian VACV isolates (VACV-Br) are divided into two groups based on their virulence in mice infections: group 1 (less virulent) and group 2 (more virulent). Poxviruses multiplication is connected to the endoplasmic reticulum (ER) at the level that viral factories produced during replication are enveloped by their membranes. Moreover, the protein folding to achieve their correct three- dimensional conformation occurs mostly in the ER lumen. As a consequence, ER can respond to disturbances caused by the accumulation of unfolded proteins, leading to adequate processing of these proteins or even programmed cell death, through the unfolded protein response (UPR) pathway. This pathway has three main sensors, which include the ATF6, IRE1 and PERK proteins. This pathway is essential in the immune response, leading to the maturation and differentiation of immune cells and the production of cytokines. Therefore, the modulation of the UPR pathway could play an important role in the immune response triggered by poxviruses infection. In this project, we investigated the effects of the infection of VACV isolates Guarani P1 virus (GP1V) and Passatempo virus (PSTV), on the activation of UPR pathways in mouse embryo fibroblasts cells (MEFs). Reporter gene assays showed us the activation of ATF6 following VACVbr infection. In one-step / multi-step growth it was not possible to observe differences in the productivity of these viruses in the absence or presence of ATF6, however, in the plaque phenotype, it is possible to observe that in ATF6-WT cells the lysis plates are smaller when compared to the plates formed in the ATF6-KO cells. Through the RFLP assay, we verified that infection by the three analyzed viruses negatively modulates to the processing of the XBP1mRNA. The evaluation of the plaque phenotype in the presence of the IRE1 kinase and Rnase domain inhibitors showed us a decrease in the VACV-Br lysis plaque in the presence of the kinase domain inhibitor. One step curves in MEF-WT and PERK-KO cells showed no difference in GP1V, PSTV, and WR virus yield, however, plaque phenotype assays showed larger lysis plates in PERK-KO cells when compared to those formed in MEF-WT cells. The one-step curves performed in the presence of the BiP inhibitor showed no difference in VACV-Br productivity, however, in the plaque phenotype, it is possible to observe smaller size lysis plates in the wells treated with the inhibitor. qPCR assays have shown that infection with GP1V, PSTV and WR viruses regulates the expression of genes responsive to the UPR pathway. In conclusion, ATF6 seems to have an important role during VACV-Br infection. The three viruses efficiently multiplied in cells deficient for the PERK sensor. The IRE1 kinase domain plays an important role in the replication of GP1V, PSTV and WR viruses.