Caracterizacion de las proteínas estructurales del torovirus equino BEV. Estudio de la inducción de apoptosis por BEV e identificacion de proteinas virales implicadas

Abstract

Toroviruses are single stranded positive RNA viruses belonging to the Coronavirus family of the Nidovirales order. They cause gastroenteritis in horses, cows, pigs and humans. These viruses have been poorly characterized, even the equine isolate BEV that is the prototype member, and the only torovirus that can be grown in cultured cells. In this work, we have expressed the BEV M, N, and HEX structural proteins through the baculovirus expression system to obtain large amounts of these proteins to produce specific antibodies in different animal species. With these tools, and with an anti-BEV polyclonal antibody that recognizes both N and S proteins, we have been able to initiate the characterization of the structural proteins of BEV. We have used a vaccinia recombinant system to express them in mammalian cells, and we have compared their behaviour when expressed alone and in the BEV infection context. We found that the N protein is phosphorylated both at Ser and Thr residues, and exhibits a cytoplasmatic localization; M is a 22 kDa protein that forms dimers, is secreted to the extracellular medium, and in the cell is localized to the ERGIC region with a luminal N-terminus and a cytoplasmic C-terminal end; and the S protein is produced as a 200 kDa precursor that is proteolitically processed to give raise to cleaved products of 75-100 kDa in cells expressing a recombinant S protein, as it occurs in both in BEV infected cells. This protein is located in the ERGIC when expressed in the BEV infection context, and in the ERGIC, cytoplasm and membrane when expressed alone. Also, we found that BEV induces apoptosis in the infected cells, and both death receptor and mitochondrial pathways are implicated. We have also studied changes in host gene expression triggered by BEV infection. We have determined that M and S structural proteins, and also N protein at a lower level, are involved in that induction, as they produce a similar effect when expressed from a vaccinia virus vector. To confirm the occurrence of apoptosis we have studied several apoptosis markers like protein synthesis inhibition, nuclei disorganization, cell cycle stage, and RNA, PARP and eIF4GI degradation. We have also performed different analyses to characterize this process by using different caspase inhibitors, bcl2 and non phosphorylable eIF2α sobreexpression, non phosphorylable eIF2α cells and Ser/ Thr kinases knock out cells. We have also observed that this phenomenon occurs in several types of cells, both by BEV infection (E. Derm and MRC5) or by BEV protein expression through vaccinia virus recombinants (HeLa, BSC40, NIH 3T3, MEF, BHK-21, 293T). Abstract I