The Study On The Degradation Of The Omega-3 Fatty Acid Transporter Mfsd2a By Zika Virus And Pathogenic Mechanisms

Zika Virus(ZIKV)is an emerging arbovirus and is in the genus Flavivirus in the Flaviviridae viral family.The main route of transmission of ZIKV to people is via the bites of infected Aedes genus of mosquitoes.Unlike any other known flavivirus,ZIKV can be spread from person to person.Nonvector arbovirus transmission has been reported to occur directly between vertebrates,from mother to child,nosocomially,by transfusion,via bone marrow or transplantation,and sexually.The recent outbreak of ZIKV in Brazil and association with fetal abnormalities including microcephaly has attracted widely attention worldwide.ZIKV can be sexually transmitted in human and passed from infected mother to their fetuses through vertical transmission.ZIKV infection during pregnancy has been linked to neonatal malformations,resulting in various degrees of congenital abnormalities,microcephaly,and even abortion,and a strong association with ZIKV and Guillain-Barre syndrome exists in adult,causing a great threaten to human health.Studies has shown that ZIKV directly infects and acrosses brain microvascular endothelial cells in the Blood-Brain Barrier(BBB).ZIKV alters vasculature integrity and results in a leaky BBB in the developing brain.Otherwise,ZIKV infection leads to the apoptosis of NPCs and massive neuronal death.The BBB is a multicellular vascular structure that separates the central nervous system from peripheral blood circulation.The core anatomical element of the BBB is the cerebral blood vessel formed by endothelial cells(ECs).The highly specialized substrate-specific transport proteins expressed in the ECs regulates the delivery of energy metabolites and essential nutrients to the brain and maintains the transport function of the BBB.Docosahexaenoic acid(DHA)is an important component of brain lipid structures and plays critical roles in maintaining the structure and function of normal brain.Because the brain cannot synthesize DHA,they must be suppied to the brain by the plasma.Mfsd2a has been recently identified as a critical component of BBB formation and integrity.Mfsd2a is a brain endothelium-enriched receptor for DHA,which is transported in the form of lysophosphatidylcholine(LPC).In addition,enrichment of DHA in the plasma membrane renders is unfavorable for the assembly of functional caveolae domains and subsequent vesicle formation.Microcephaly syndrome was recently shown to be caused by inactivating mutations in Mfsd2a,the severity of the syndrome correlating with the degree of functional inaction of the Mfsd2a protein.Mice lacking Mfsd2a show brain DHA deficits and develop BBB breakdown.Although a large number of researches have been devoted,the pathogenic mechanism of ZIKV has not been folly elucidation,especially with respect to vascular brain microenvironment changes,and it remains unclear whether ZIKV infection has affection in Mfsd2a expression and vascular brain microenvironment changes.In view of the correlation between Mfsd2a deletion and microcephaly,the study systematically analyzed the regulatory effect of ZIKV infection carried out in-depth functional analysis.To investigate whether the Mfsd2a expression level changes during ZIKV infection,we established a cell culture model of ZIKV-infected primary hBMECs and ZIKV-infected neonatal mice,we found that endogenous Mfsd2a protein was significantly downregulated after ZIKV infection.However,Mfsd2a mRNA levels were unaltered following infection.To further dissert the mechanism of Mfsd2a inhibition,we cotransfected Mfsd2a along with individual ZIKV-encoded proteins into HEK293T cells.Interestingly,ZIKV envelope(E)protein,but not other viral proteins,disrupted Mfsd2a expression.To identify specific pathways involved in Mfsd2a degradation,distinct pathway inhibitors were used for a screening experiment,MG 132 was the only tested inhibitor preventing ZIKV E from degrading Mfsd2a,suggesting that the proteasome degradation pathway plays a critical role during this process.Furthermore,the in vivo binding between ZIKV E and Mfsd2a was demonstrated by coimmunoprecipitation in the presence of MG132.We found that K3R,K46R,K296R and K436R mutants showed significant resistance against ZIKV E-mediated degradation.Moreover,ZIKV E transgene suppressed the brain endothelial Mfsd2a expression and resulted in a similar but mild microcephaly phenotype compared with ZIKV-infected mice.These data indicate that ZIKV E specifically interacts with the orphaned transporter Mfsd2a and promotes the ubiquitination of Mfsd2a for proteasome-dependent degradation.The degradation of Mfsd2a by ZIKV E may be one of the main causes for ZIKV infection related microcephaly.The neurovascular unit is responsible for brain microenvironment maintenance and a continuous supply of nutrients,in which an influx of DHA is essential for normal brain development and cognitive function.Brain uptake of DHA is mediated by Mfsd2a only in the form of esters with LPCs.We next sought to evaluate the LPC uptake efficiency by using the LPC fluorescent derivative named TopFluor LPC.Infection of ZIKV isolates or tansfection of ZIKV E almost abolished LPC uptake in hBMECs and Mfsd2a-overexpressing A549 cells.Using a lipidomic analysis,we found that the total level of DHA was significantly reduced in ZIKV-infected cells compared to that in the mock-infection group.Supplementation with DHA or OA(oleic acid)increased the brain Mfsd2a level and suppressed ZIKV RNA copies and slightly protected animals against ZIKV-induced phenotypes.In addition,the administration of DHA or OA was a positive factor for neuron survival during ZIKV infection.Together,these results suggest that Mfsd2a was significantly disrupted during ZIKV infection or overexpressed virus E protein,and therefore the Mfsd2a-mediated LPC uptake was blocked and lipid homeostasis was impaired.Here we found a pivotal role for Mfsd2a in the pathogenesis of ZIKV.Infection of ZIKV disrupted Mfsd2a in both cultured primary hBMECs and the neonatal mouse brain.ZIKV envelope(E)protein specifically interacted with Mfsd2a and promoted Mfsd2a polyubiquitination for proteasome-dependent degradation.ZIKV E transgene partially recapitulated the microeephaly phenotype.Infection with ZIKV or ectopic expression of ZIKV E impaired Mfsd2a-mediated DHA uptake into cells.A lipidomic analysis revealed obvious differences in hBMEC lipid signatures after ZIKV infection.Intriguingly,supplementation with DHA by intraperitoneal injection rescued ZIKV-caused growth restriction and microcephaly.Our findings suggest endothelial Mfsd2a as an important pathogenic mediator and supplementation with DHA as a potential therapeutic option for ZIKV infection.