Human cytomegalovirus (HCMV) is a virus infecting the majority of adults worldwide. In healthy individuals, a strong immune response to HCMV is able to limit and contain the spread of the disease . HCMV can infect a remarkably broad cell range within its host. The broad cell tropism of HCMV may reflect the abundance of distinct glycoprotein complexes in the virion envelope . The core machinery for Herpesvirus entry comprises three highly conserved viral glycoproteins, glycoprotein B (gB), glycoprotein H (gH), and glycoprotein L (gL) . In addition to gB and gH/gL, most Herpesviruses encode additional glycoproteins that are able to interact with gH/gL. For HCMV, this addition consist of the glycoprotein gO, to form gH/gL/gO complex, or the trimer UL128/UL130/UL131A (referred as “ULs”), to form a pentameric structure often designated as “Pentamer”. Viral entry into fibroblast or epithelial/endothelial and lymphoid cells relies on the presence of gH/gL/gO or Pentamer respectively [35, 40]. In an in vitro system, specific cysteines have been identified to stabilize these complexes and impairment of disulfide bonds formation abolishes complexes maturation and cellular trafficking . Here we addressed the relevance of these disulfide bonds in the formation of HCMV entry complexes and on the infectivity of point mutated viruses. To this purpose, four recombinant Cys-mutated viruses, generated through mutagenesis of a Bacterial Artificial Chromosome (BAC) containing the entire genome of HCMV TR strain, were analysed for viral tropism on three different cell types. We also checked by Western blot the content of the pentameric proteins expressed by these mutants both in the extracts of infected fibroblasts and monocytic cells (HFF and THP-1, respectively) and in virions produced by infection of human fibroblasts. Surprisingly, results from our analysis showed that mutation on two specific cysteines involving gL disulfide bonds to gO or UL128 and to gH resulted in the loss of intracellular gL or expression level under the detection power of Wb. Two other Cys mutated viruses showed no differences in the levels of viral structural proteins compared to wt. These results suggest that the impairment of the disulfide bond involving binding of gL to UL128 or gO and gL to gH, cause instability of the gL protein with loss or reduced ability to form higher order complexes and likely cellular degradation. However, our results show that infectious viruses can achieve a complete life cycle in absence of a crucial protein like as gL but it also raise the question of which pattern of factors, likely interacting with gH, are necessary as “surrogate” gL.
|Titolo:||Functional and structural characterization of HCMV complexes by dissecting molecular interactions|
|Data di discussione:||23-feb-2018|
|Appartiene alla tipologia:||07a Tesi di Dottorato|