Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca(2+) concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH's antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.

Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.