HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.

Background: The Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis.

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import. M ost proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes and must therefore be imported across the outer and inner mitochondrial membranes. Translocation across the inner membrane occurs through the inner membrane channel and is driven by the membrane potential and an import motor (1-3). Three critical components of this motor are the major Hsp70 molecular chaperone of mitochondria (mtHsp70; Ssc1 in yeast); the peripheral inner membrane protein Tim44, which tethers Ssc1 to the import channel; and the nucleotide release factor for Ssc1, Mge1. Multiple cycles of Ssc1 binding to and release from translocating polypeptide, driving the import process, are required for import of proteins into the mitochondrial matrix (1-3).Ssc1, like other Hsp70s, contains a C-terminal domain that binds short segments of unfolded polypeptides rich in hydrophobic amino acids (4, 5). The N-terminal ATPase domain regulates this binding through interaction with adenine nucleotides. In turn, binding of the peptide segment to the C-terminal domain stimulates the ATPase activity of the N terminus. In the ATP-bound state, interaction with peptide substrate is unstable, with very fast on and off rates. In the ADP-bound state, the interaction is relatively stable, with slow on and off rates. Therefore, it is thought that the ATP-bound form of Hsp70 initiates interaction with polypeptide substrate, which is then stabilized by the hydrolysis of ATP. Exchange of ATP for bound ADP results in the release of peptide, thus completing the cycle.Hsp70s rarely, if ever, function independently. Rather, they function with cochaperones. J proteins, named because of the presence of the signature J domain that interacts with the ATPase domain, stimulate Hsp70's ATPase activity, thus facilitating interaction with substrate polypeptides (6, 7). This activity is a critical feature of J...

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