Search

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Human immunodeficiency virus type 1 (HIV-1) can establish latent infection following provirus integration into the host genome. NF-kappaB plays a critical role in activation of HIV-1 gene expression by cytokines and other stimuli, but the signal transduction pathways that regulate the switch from latent to productive infection have not been defined. Here, we show that ERK1/ERK2 mitogen-activated protein kinase (MAPK) plays a central role in linking signals at the cell surface to activation of HIV-1 gene expression in latently infected cells. MAPK was activated by cytokines and phorbol 12-myristate 13-acetate in latently infected U1 cells. The induction of HIV-1 expression by these stimuli was inhibited by PD98059 and U0126, which are specific inhibitors of MAPK activation. Studies using constitutively active MEK or Raf kinase mutants demonstrated that MAPK activates the HIV-1 long terminal repeat (LTR) through the NF-kappaB sites. Most HIV-1 inducers activated NF-kappaB via a MAPK-independent pathway, indicating that activation of NF-kappaB is not sufficient to explain the activation of HIV-1 gene expression by MAPK. In contrast, all of the stimuli activated AP-1 via a MAPK-dependent pathway. NF-kappaB and AP-1 components c-Fos and c-Jun were shown to physically associate by yeast two-hybrid assays and electrophoretic mobility shift assays. Coexpression of NF-kappaB and c-Fos or c-Jun synergistically transactivated the HIV-1 LTR through the NF-kappaB sites. These studies suggest that MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 LTR. These results define a mechanism for signal-dependent activation of HIV-1 replication in latently infected cells and suggest potential therapeutic strategies for unmasking latent reservoirs of HIV-1.

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in virus replication and infectivity. Here we show that Vif is phosphorylated and regulated by p44/42 mitogen-activated protein kinase (MAPK). Vif phosphorylation by MAPK was demonstrated in vitro as well as in vivo and was shown to occur on serine and threonine residues. Two-dimensional tryptic phosphopeptide mapping indicated that Vif is phosphorylated by MAPK on the same sites in vitro and in vivo. Radioactive peptide sequencing identified two phosphorylation sites, Thr96 and Ser165. These phosphorylation sites do not correspond to the known optimum consensus sequences for phosphorylation by MAPK (PX(S/T)P) nor to the minimum consensus sequence ((S/T)P), indicating that MAPK can phosphorylate proteins at sites other than those containing the PX(S/T)P or (S/T)P motifs. Synthetic Vif peptides corresponding to the local sequences of the phosphorylation sites were not phosphorylated by MAPK, suggesting that recognition of these sites by MAPK is likely to require structural determinants outside the phosphorylation site. Mutations of the Thr96 site, which is conserved among Vif sequences from HIV-1, HIV-2, and SIV, resulted in significant loss of Vif activity and inhibition of HIV-1 replication. These results suggest that MAPK plays a direct role in regulating HIV-1 replication and infectivity by phosphorylating Vif and identify a novel mechanism for activation of HIV-1 replication by mitogens and other extracellular stimuli.