Functional Assignment Of Multiple ESCRT-? Homologs In Cell Division And Virus Budding In Sulfolobus Islandicus

Cell division is a fundamental process in all cellular life forms.In bacteria,cell division is mainly mediated by FtsZ and FtsA(MreB),which are the eukaryotic homologs of tubulin and actin,respectively.FtsZ forms a constricting ring structure(Z-ring)with the help of FtsA at the nascent division site,and then more than 20 proteins are recruited at the Z-ring and form the divisome.The Z-ring can also provide a constricting force and finally one parental cell divides into two sister cells.In eukarya,cell division starts with actin and myosin that form a constricting ring.The parental cell will divide into two parts with the constriction of the constricting ring,but the two sister cells are still connected together by the intercellular bridge and the mid-body structures.The ESCRT-? complex is then recruited at the mid-body structure and cut the membrane adjacent the mid-body and finally the two sister cells separate from each other.In archaea,it has been indicated that at least three distinct cell division modes exist:the FtsZ-based bacterial mode in Euryarchaeota and Nanoarchaeota;the ESCRT-? machinery based eukaryotic-like mode in TACK(Thaumarchaeota,Aigarchaeota Crenarchaeota and Korarchaeota),which include the majority of Crenarchaeota,and Asgard(Lokiarchaeota,Thorarchaeota,Odinarchaeota and Heimdallarchaeote);and a putative novel crenactin-based mode in Thermoproteales of Crenarchaeota.Sul.folobus uses the ESCRT-? machinery for cell division.It comprises three proteins:CdvA(an archaea-specific protein)as well as the eukaryotic-like ESCRT-?(CdvB)and Vps4(CdvC)proteins.Similar to its eukaryotic counterparts,Sulfolobus contains several ESCRT-? proteins.In addition to ESCRT-?,Sulfolobus encode three other ESCRT-? homologs termed ESCRT-?-1(CdvBl),ESCRT-?-2(Cdv-B2)and ESCRT-?-3(CdvB3).Whether these three ESCRT-? homologs are involved in cell division and the specific roles of the cell division proteins including CdvA and ESCRT-? during cell division are still unknown.In this thesis,we analyzed the function of the ESCRT-? homologs and determined the functional localization of the proteins during the process of cell division in Sulfolobus islandicus,by means of genetics,cell biology and so on.We found that ESCRT-?-1 and ESCRT-?-2 localized at mid-cell forming band-like(ring-like)structures,and the structures constricted with the proceeding of cell division.These indicate that ESCRT-?-1 and ESCRT-?-2 play direct roles in cell division.By contrast,ESCRT-?-3 dispersed relatively evenly in the cell,indicating that ESCRT-?-3 does not have a role in cell division.In concert with their roles in cell division,escrt-?-1 and escrt-?-2 were indispensable for cell viability,while escrt-?-3 could be deleted and the deletion did not have any obvious effects on cell growth and morphology.We determined the interaction network of the cell division proteins in Sulfolobus islandicus.Based on the interaction network and the secondary structures of the proteins,we constructed the C-terminal truncated mutants of these proteins,which impeded their interactions.By over-expressing the C-terminal truncated mutants of these proteins in S.islandicus cells,we revealed that the division proteins played important roles at different stages of cell division.These results are of great significance to elucidate the specific processes of cell division in Sulfolobus.Notably,in cells over-expressing the C-terminal truncated mutant of ESCRT-?-2,we observed eukaryotic-like intercellular bridge and mid-body structures,which were observed in archaea for the first time.These results further prove that many archaea like Sulfolobus probably use a eukaryotic-like mechanism for cell division and add a new common featurebetween archaea and Eukarya.In eukaryotes,except for their roles in cell division,the ESCRT-? proteins also participate in membrane-enveloped virus budding.Sulfolobus tengchongensis spindle virus 2(STSV2),is a membrane-enveloped sigle-tailed fusiform virus isolated fromthe Tengchong hot spring of China's Yunnan province.Itis an ideal model virus for archaeal virus-host studies,since it can be stably cultured over long periods with laboratory strains of Sulfolobus,and no evidence has been found that it can lyse host cells under different stress conditions.In the work of-this thesis,we observed budding phenomenon in STSV2 infected S.islandicus REY15A cells,and found that ESCRT-?-3 could localize at the budding sites.However,we could not observe the budding structures in STSV2 inf-ected ? escrt-?-3 cells.These results indicate that ESCRT-?-3 plays an important role in the viral budding process.In addition,we observed ESCRT-?-1 and ESCRT-?-2 could also localize at the budding site,indicating that ESCRT-?-1 and ESCRT-?-2 may probably play roles in the budding process too.In eukaryotes,the virus of HIV-1,influenza and Ebola also generally use the budding process for release from the host cells,and ESCRT-?proteins play a pivotal role during the process.Our results reveal that STSV2 uses a mechanism that ESCRT-? proteins play important roles in viral budding,which is similar to its eukaryotic counterparts.Now,more and more data from comparative genomics and phylogentic analysis support for the hypothesis that eukarya origins from the archaea,but the biggest problem in it is its lack of experiment data.This hypothesis is indeed supported by our experiment results,which may have significant implication in evolution.Our studies may provide insight for the study of eukaryotic cell division and the budding process of membrane-enveloped virus and may also offer some help for the therapy of human viruses,such as HIV-1,influenza,and Ebola.