Mechanism Research On The Interaction Between HIV Vif And T Cell Differentiation Factor CBF?

HIV(human immunodeficiency virus)is an RNA retrovirus with 9.2~9.7kb genes,that encoding three structural proteins and six accessory proteins.After infection,HIV targets and destroys the immune system,leading to the development of AIDS(acquired immune deficiency syndrome),carcinoma genesis,and opportunistic infections.HIV Vif(viral infectious factor)protein,192 amino acids in length,is an important accessory HIV protein.Vif protein has two main functions: one is the degradation of host cell antiviral factors(such as the APOBEC3 family proteins),to break through host innate immune defenses;the other is to suppress the growth of infected cell by inducing G2/M cell cycle arrest.These features greatly improve the replication of the HIV infection.Based on the interaction between HIV Vif and the T-cell differentiation factor CBF?,this thesis focused on the importance of CBF? upon Vif functions and subsequently HIV replication,the molecular mechanism of Vif-CBF? interaction,and the search for inhibitory approach against Vif-CBF? interaction.And we found that: 1.CBF? is required for HIV Vif functions.First,we focused on the Vif-CBF? interaction and its mechanism.To confirm that CBF? is important to Vif,known functions of Vif were tested in HEK293 T and H9 cells where endogenous CBF? expression was knocked down.As shown in Results,in the absence of CBF?,Vif could not induce the degradation of APOBEC3 proteins or arrest host cells at the G2/M phage.Long-term infection experiments proved that HIV could not replicate effectively without CBF? in H9 cells.Further investigation indicated that,CBF? was required for Vif to bind Cullin5 and hijack endogenous ubiquitination complex.Thus,CBF? is a crucial binding factor for Vif,to promote Vif functions and ensure the replication of HIV in non-permissive cells.2.Vif and RUNX probably interact with CBF? through different interfacesCBF? interacts with endogenous DNA binding factors termed RUNX,to form heterodimers that are critical for the development and regulation of human immune system.In this study,we investigated a variety of CBF? mutants on their ability of interacting with Vif/RUNX,and their effects on Vif/RUNX function.We observed that different regions on CBF? were in charge of interacting Vif or RUNX,indicating that Vif and RUNX interact with CBF? through different interfaces.This suggested that it is possible to develop approaches to sabotage Vif-CBF? without compromising RUNX-CBF? interaction,thus revealing a new concept for the development of novel anti-HIV drugs.3.Discovering a CBF?-based protein that suppresses Vif but not RUNX functions or cell viabilityTo further prove the concept above was practicable,based on the interaction between CBF?,Vif,and RUNX,we searched for CBF? mutants that could suppress Vif but not RUNX.As shown in Results,we found a CBF? fusion protein termed CBF?-SMMHC ?C95,which suppresses Vif functions and HIV replication even with the presence of wild type CBF?.On the other hand,?C95 does not affect the activity of RUNX,nor the viability of cells in vitro.The discovery of ?C95 not only confirms that the development of novel anti-HIV drugs targeting Vif-CBF? interaction is practically practicable,but also proves that the interaction between Vif and CBF? is essential for Vif functions and HIV replication.In conclusion,this paper revealed detailed molecular mechanism for VifCBF? interaction,the importance of CBF? upon Vif functions and HIV replication,the relationship between Vif-CBF? and RUNX-CBF? heterodimers,and the feasibility for the concept of new anti-HIV drugs targeting Vif-CBF? interaction.Such information on one hand perfects the understanding on the molecular mechanism of Vif functions;on the other hand,it also provides new target for the development of novel anti-HIV drugs,thus being important for the future treatment and/or prevention of AIDS.