| The viral infectivity factor (Vif), one of the six HIV-1 accessory protein, isabsolutely necessary for productive infection in primary CD4-positive Tlymphocytes and macrophages. It is believed to act during the late stages of virusassembly by enabling the establishment of integrated provirus in new target cells.It is a 23-kDa basic protein, encoded by 192 a.a . The gene, discovered in themid 1980s,was at first entitle sor. The gene product was later renamed Vif for itsfunction. Some selected cell type infected by Vif-mutant virions can not produceinfectious virus particles. The producer cells (e.g., H9 and CEM T cell lines)were described as 'non-permissive'. Other selected cell lines , such as SupT1,293T e.g., did not seem to require Vif for producing infectious and replicatingvirions, and were called 'permissive'. Since Vif-mutant virions show severelyimpaired infectivity, Vif must regulate one or more of the molecules found invirions. In 1998, two groups demonstrated that there seemed to be anendogenous cellular cofactor inhibitor of HIV-1, which was overcome by theviral Vif protein. This factor appeared to alter Vif mutant HIV-1 at the late stagesof the viral life cycle in non –permissive producer cells. Then the factor wasfound as CEM15, which was renamed APOBEC-3G .APOBEC-3G, the antiviral host factor, belongs to a family of proteins thathave cytidine deaminase activity. In the absence of Vif, APOBEC-3G induces thedeamination of cytosines incorporated in the minus single-strand of the viralDNA during the reverse transcription. Vif-mutant HIV-1 viruses produced in thepresence of APOBEC3G undergo hypermutations in newly synthesized viralDNA, presumably due to C-to-U modification during minus-strand viral DNAsynthesis. The HIV-1 Vif protein counteracts APOBEC-3G by decreasing itspackaging into virions.Moreover ,Vif descrease the intracellular level of APOBEC-3G in theinfected cells probably by several independent mechanisms. One of themimplicates the poly-ubiquitination of APOBEC-3G induced by Vif and itsdegradation by the proteasome. In 2003,YU e.g. reported that Vif interacts withcellular protein Cul5,elongin B,and Rbx I to form an Skp1-cullin-F-box(SCF)-like complex, allowing Vif to interact with APOBEC-3G and induce itsubiquitination and degradation.In this study, we sought to approve the interactions between every twoproteins of Vif-Cul5 –SCF-like complex. We used the Mammalian Two-Hybridesystem ,a powerful methods for detecting protein:protein interactions in vivo.There are two vectors of this system, pBIND (with orf of GAL4 fusionprotein )and pACT(with orf of VP16 fusion protein). Wo respectively cloned thecDNA of Vif, CEM15, Cul5, ElonginB, ElonginC, and Rbx I into the two vectors,inorder to make them fused to GAL4 or VP16, both of which are transcriptionactivation factor. Every two kinds of clones co-transfected COS 7 cells with pG5luc vector of firefly luciferase gene. Then we harvested the cell and investigatedprotein : protein interaction by evaluating the expression level of luciferase.The results show that when there is no other protein involved in ,the Vifprotein interacts with ElonginB, but not with ElonginC or not enough strongly,despite they do in the E3 complex. So we hypothesize that Vif's interaction withElonginC in vivo must be accelerated by someother protein, which just maybethe ElonginB.We observed the interaction between Vif and CEM15/APOBEC-3G for thefirst time. The result indicates that Vif interact with APOBEC-3G directly, noneed for others linking protein.We also screened the ligands of Vif protein by 12-mer phage displaypeptide library. For the poor solubility of Vif, we preparation Vif–ΔN protein astarget protein,which is without 28 a.a of N-end (most are hydrophobic). After 3-4rounds of screening and affinity test by ELISA, we got selected individual clone.We blasted their sequence for the conserved part. That provide new project forthe design of Vif small peptide inhibitor.
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