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In Varicella-zoster virus (VZV) the most expressed protein during infection is ORF9p. This protein is conserved in all the Alphaherpesvirus and is an essential tegument protein. ORF9p interacts with IE62 the major VZV transactivator which is also part of the tegument although the mechanisms leading to its incorporation into the particle are poorly understood. Considering that both proteins are present in high quantity in the viral tegument, it is suggested that one of the ORF9p functions is the recruitment of IE62 in order to be incorporated into the tegument (Cilloniz et al., 2007). Previous studies done in the laboratory prove that ORF9p plays an important role during secondary envelopment and viral particle formation. It is suggested that this main function is regulated by its phosphorylation and the interaction with other viral proteins (Riva et al., 2013). The first goal of this work is to determine if the phosphorylation of ORF9p is important for the interaction with IE62. The second aim is to determine the responsible site of ORF9p for the interaction with IE62 and glycoprotein gE. The role of ORF9p phosphorylation in the interaction with IE62 has been analysed using different ORF9p mutants lacking a phosphorylation by the ORF47 viral kinase. On one hand, we have demonstrated that the interaction between ORF9p and IE62 does not depend on the ORF9p phosphorylation level. On the other hand, we observe a co-localisation of both proteins accumulate in subcellular structures that accumulates in the vicinity of the nucleus and whose nature has still to be determined. More broadly, using mutants unable to express ORF47p and/or ORF66p, the two ser/thr kinases encoded by the virus or-phosphatase treatments, we showed that nor the phosphorylation of ORF9p or IE62 was important for their interaction. In a second part, we evaluate the importance of an WW domain present ORF9p sequence and shown to be important for HSV-1 VP22 (ORF9p homologue) interaction with VP16 the major transactivator. ORF9p punctual mutants were created by mutating the two tryptophan and the phenylalanine described to be important in HSV-1. Interestingly, F181A and W201F mutations present a loss of infection over time. Therefore, these two amino acids are perhaps important for the interaction with IE62 and gE. These observations confirm that nor the phosphorylation of ORF9p and IE62 or the amino acids F181 and W201 of ORF9p, located in a putative WW domain are not important for the interaction between these 2 tegument proteins.
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Human cytomegalovirus (hCMV) is a major cause of congenital abnormalities in foetuses worldwide. These affected infants typically develop neurological sequelae, and present with dysfunctions in brain development that may lead to either severe cognitive impairment or hearing loss. The vertical transmission of hCMV from mother to foetus is primarily the result of a primary infection acquired by the mother during pregnancy. In addition, the severity of brain abnormalities has been reported to be directly proportional to the precedency of infection during gestation. Whilst animal (in particular murine) models of congenital hCMV have been developed to study the pathophysiological mechanisms underlying this disease; however, none of the previously reported studies modelled the congenital hCMV infection during the first trimester of pregnancy. Thus, the current literature is limited from further insights into hCMV-induced pathophysiological mechanisms early during pregnancy. Therefore, we decided to (develop / optimise / validate) a mouse model that phenocopies, as closely as possible, the congenital hCMV infection acquired during the first trimester of pregnancy. For this purpose, we injected the murine cytomegalovirus (mCMV) directly into the placental labyrinth of each murine embryo at embryonic day 10.5 (E10.5), whose stage of brain development corresponds to a human foetus during the first trimester. We then monitored the effects of mCMV infection on the health of pregnant mice, as well as the development of murine embryos and pups. We report findings that demonstrate mCMV infection of murine embryos via intraplacental injection at E10.5 shares similarities with congenital hCMV infection. Despite the phenocopy of previously reported human and murine signs and symptoms in our current animal model: health of pregnant mice, microcephaly and microencephaly in infected embryos and pups, recruitment of microglial cells in the embryonic brains, we intend to proceed with detailed spatio-temporal histo-cytologcal analyses to further validate our murine CMV model. In conclusion, we demonstrate a novel mouse model for a more comprehensive study of the pathophysiological mechanisms underlying congenital hCMV infection.
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Role of the Dynamin related protein 1 Drp1 in the regulation of Vascular Endothelial Growth Factor Receptor VEGFR2 signaling
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