Mitochondrial RNAs in Trypanosoma brucei undergo posttranscriptional RNA editing and polyadenylation. We previously showed that polyadenylation stimulates turnover of unedited RNAs. Here, we investigated the role of polyadenylation in decay of edited RPS12 RNA. In in vitro turnover assays, nonadenylated fully edited RNA degrades significantly faster than its unedited counterpart. Rapid turnover of nonadenylated RNA is facilitated by editing at just six editing sites. Surprisingly, in direct contrast to unedited RNA, turnover of fully edited RNA is dramatically slowed by addition of a poly(A) 20 tail. The same minimal edited sequence that stimulates decay of nonadenylated RNA is sufficient to switch the poly(A) tail from a destabilizing to a stabilizing element. Both nucleotide composition and length of the 3 extension are important for stabilization of edited RNA. Titration of poly(A) into RNA degradation reactions has no effect on turnover of polyadenylated edited RNA. These results suggest the presence of a protective protein(s) that simultaneously recognizes the poly(A) tail and small edited element and which blocks the action of a 3-5 exonuclease. This study provides the first evidence for opposing effects of polyadenylation on RNA stability within a single organelle and suggests a novel and unique regulation of RNA turnover in this system.Trypanosoma brucei is a unicellular, flagellated, parasitic protozoan that causes African sleeping sickness in humans and nagana in domestic livestock. It contains a single large mitochondrion, whose gene expression and metabolic activity are regulated throughout its life cycle. Procyclic (insect) form parasites contain a fully functional mitochondrion that carries out oxidative phosphorylation (3,29,42). In contrast, bloodstream (mammalian) form trypanosomes exhibit a high rate of glycolysis and contain an underdeveloped mitochondrion (3,29,42). Similar to other eukaryotes, the genes for the mitochondrial proteins are encoded both in the nuclear and mitochondrial genomes.Since transcription of T. brucei mitochondrial DNA generates polycistronic transcripts (14,19,22,33), posttranscriptional mechanisms of gene expression and their regulation are of prime importance. Maturation of mitochondrial RNAs requires several posttranscriptional processing steps, including precursor cleavage, RNA editing, and polyadenylation. Polycistronic precursors must be endonucleolytically cleaved and possibly exonucleolytically trimmed to create the correct 5Ј and 3Ј ends of individual monocistronic RNAs. In addition, 12 of the 18 transcripts in T. brucei mitochondria require editing, which involves specific insertion and deletion of uridine residues, to create translatable RNAs (for recent reviews see references 27, 37, 38, and 40). mRNAs are also extended at their 3Ј ends by polyadenylation. The length of poly(A) tail has been found to correlate with the edited status of the RNA. While unedited RNAs contain either no or short (ϳ20 nucleotides [nt]) poly(A) tails, edited RNAs, including partia...

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.

Summary Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121-125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.