The role of Epstein-Barr virus stable intronic sequence-RNAs in early B Cell infection

Epstein-Barr virus (EBV) is a gamma-herpesvirus that infects epithelial cells and naïve B cells. With a prevalence of about 95% in the world population, it is generally acquired during childhood or adolescence and is responsible for a non-threatening clinical condition called infectious mononucleosis. However, particularly in the immunosuppressed this virus is implicated in the pathology of lymphomas and some carcinomas. The presence of EBVencoded stable intronic sequence RNAs in EBV-positive cell lines was detected for the first time in 2013 and their role in the EBV’s life cycle is not yet known. Here is reported the generation of the first virus knock-out virus for sisRNA-1 and the preliminary study of the virus’ behaviour in B cell infection. Evidence found by monitoring cell proliferation through microscopy and FACS analysis shows that knocking out the intron that encodes sisRNA-1 causes proliferation impairment by day 8 after B cell infection, but doesn’t impede later proliferation and outgrowth of LCLs. Viral transcription of LMP1 and Cp vs Wp promoter usage was analysed as well as viral protein expression of the EBNAs but no significant differences between WT and sis1KO where found. It remains to be determined if the effects observed where in fact a consequence of the lack of sisRNA-1 or due to the disruption of BHRF1 splicing that we hypothesised to be a possible consequence of deleting sisRNA-1.O vírus Epstein-Barr é um gamma-herpesvirus que infecta células epiteliais e células B “naive”. Com uma prevalência de cerca de 95% na população mundial, é geralmente adquirido durante a infância ou adolescência, sendo responsável por uma condição clínica, designada mononucleose infeciosa, que não ameaça a vida. No entanto, particularmente em indivíduos imunodeprimidos este vírus está implicado no aparecimento de linfomas e de alguns carcinomas. A presença de “stable intronic sequence RNAs” (sisRNAs) codificados pelo EBV foi detectada em linhas celulares EBV-positivas pela primeira vez em 2013 e a sua função no ciclo de vida do vírus tem sido até agora desconhecida. Aqui é reportada a produção do primeiro vírus knock-out para o sisRNA-1 e o estudo preliminar do seu comportamento na infecção de células B. Os resultados obtidos a partir da monitorização da proliferação celular e análises de FACS revelaram que a delecção do intrão codificado pelo sisRNA-1 causa um atraso na proliferação celular no dia 8-pós infecção de células B, mas não impede a proliferação mais tardia e a formação de LCLs. A transcrição de genes virais como o LMP1 e a utilização dos promotores Cp vs Wp, bem como a expressão de proteínas virais foram analisadas, mas nenhuma diferença significativa foi encontrada entre os vírus WT e o sis1KO. Permanece por determinar se os efeitos observados são de facto uma consequência do sisRNA-1 ou da disrupção do “splicing” de BHRF1 que consideramos ser uma consequência possível da delecção do sisRNA-1

A role for myotubularins in vaccinia virus egress

Vaccinia virus (VACV) generates multiple distinct forms of infectious virus. Intracellular mature virus (IMV) particles are surrounded by a single lipid membrane and are mostly released upon lysis of the infected cell. Some IMVs are wrapped by an additional double membrane to form intracellular enveloped virus (IEV), also called wrapped virus (WV). IEVs are transported on microtubules to the cell surface where the outer membrane fuses with the plasma membrane (PM) to expose a double enveloped virion outside the cell. Extracellular enveloped virus (EEV) and CEV are surrounded by two membranes and are morphologically indistinguishable, but whereas EEV are released from the cell to mediate long range virus spread, CEV are retained on the cell surface and induce the formation of actin tails to propel virions to neighbouring cells. EEV and CEV are essential for efficient virus spread within the host. This thesis concerns viral and cellular factors that influence transport of IEV particles from the site of wrapping to the cell membrane. For transport to the cell surface, IEVs rely on active microtubular transport, mediated by the cellular motor protein kinesin-I and the viral proteins E2, F12, which form a complex, and A36. While some of the players in this process are known, other aspects remain elusive, such as the mechanism by which E2/F12 are recruited to IEV, because they lack transmembrane regions. To study factors affecting IEV egress, a proteomic study was performed with A36, E2 and F12 as bait, and many potential interaction partners were identified. Amongst them, two cellular phosphoinositide (PI) phosphatases, myotubularin related protein (MTMR) 1 and su(var), enhancer of zeste, trithorax (SET)-binding factor (SBF) 1 were identified as major hits for the joint interactome of E2 and F12. This thesis has investigated the potential role of these and other cellular proteins in IEV egress. MTMR1 and SBF1 both co-precipitated with E2 and F12 only when both viral proteins were present and required the full length E2 protein. The co-purification of the catalytically active myotubularin MTMR1 with the E2/F12 complex required the inactive SBF1. Knockout (KO) of SBF1 was found to reduce virus spread, EEV titres and CEV numbers, confirming its involvement in IEV morphogenesis or egress. Interestingly, KO of MTMR1 did not cause a phenotype, likely because of functional redundancy between different myotubularins (MTMs). Indeed, when MTMR1 and MTMR2 were both deleted, a similar phenotype to that of SBF1 KO cells was observed. While a level of redundancy seems to exist between the active forms, the KO of the inactive myotubularin SBF2 in SBF1 KO cells did not further the defect in spread. The defect in spread observed in both SBF1 KO and MTMR1/2 double KO cells was dependant on the presence of F12/E2 and resulted from an accumulation of IEV at the sites of wrapping, partially recapitulating the phenotype observed in infections with viruses lacking E2 or F12. Furthermore, the contribution of myotubularins to IEV egress depended on the presence of the CC domain of SBF1 and MTMR1, that allows them to heterodimerise, their membrane recruitment domains (RID) and the phosphoinositide phosphatase activity of MTMR1. All together, these data show VACV hijacks phosphoinositide signalling through the recruitment of myotubularins to promote IEV egress and supports endosomes as a source of membranes for wrapping of IEVs. Finally, we also identified a role for PI3P regulation in IMV morphogenesis

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

© 2020 The Authors.The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.Amorim et al. find that Nog2 is expressed in the zebrafish notochord by the action of a tissue-specific enhancer, and it diffuses to the pancreatic domain and controls its size. The identification of Nog enhancers in other vertebrate lineages suggests a conserved mechanism for pancreas development in vertebrates.This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant ERC2015StG680156ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Investigator position (grant IF/00654/2013). J.T. and J.P.A. are PhD fellows from FCT (grant SFRH/BD/126467/2016 to J.T. and grant SFRH/BD/145110/2019 to J.P.A.). M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation program (grant 668981). The authors acknowledge the support of i3S Scientific Platform Advanced Light Microscopy, a member of the national infrastructure PPBI (Portuguese Platform of BioImaging) (supported by POCI010145FEDER022122)