Structural consequences of cyclophilin A binding on maturational refolding in human immunodeficiency virus type 1 capsid protein.

While several cellular proteins are incorporated in the human immunodeficiency virus type 1 virion, cyclophilin (CyP) A is the only one whose absence has been demonstrated to impair infectivity. Incorporation of the cytosolic protein results from interaction with a highly exposed Pro-rich loop in the N-terminal region of the capsid (CA) domain of the precursor polyprotein, Pr55(Gag). Even when prevented from interacting with CyP A, Pr55Gag still forms particles that proceed to mature into morphologically wild-type virions, suggesting that CyP A influences a postassembly event. The nature of this CyP A influence has yet to be elucidated. Here, we show that while CyP A binds both Gag and mature CA proteins, the two binding interactions are actually different. Tryptophan 121 (W121) in CyP A distinguished the two proteins: a phenylalanine substitution (W121F) impaired binding of mature CA protein but not of Gag. This indicates the occurrence of a maturation-dependent switch in the conformation of the Pro-rich loop. A structural consequence of Gag binding to CyP A was to block this maturational refolding, resulting in a 24-kDa CA protein retaining the immature Pro-rich loop conformation. Using trypsin as a structure probe, we demonstrate that the conformation of the C-terminal region in mature CA is also a product of maturational refolding. Binding to wild-type CyP A altered this conformation, as indicated by a reduction in the accessibility of Cys residue(s) in the region to chemical modification. Hence, the end result of binding to CyP A, whether the Pro-rich loop is in the context of Gag or mature CA protein, is a structurally modified mature CA protein. The postassembly role of CyP A may be mediated through these modified mature CA proteins.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

The major homology region of the HIV-1 gag precursor influences membrane affinity.

Assembly of retroviruses, including HIV-1, involves movement of newly synthesized viral proteins and RNA to the plasma membranes of host cells. The major homology region (MHR, aa 285-304), a highly conserved sequence in the capsid domain of the HIV-1 Gag precursor polyprotein, plays a critical, but unknown, role in infectious particle assembly. Mutations of invariant residues in the sequence have pleiotropic effects: Mutation of Gln287 blocks viral assembly while mutation of Arg299 permits assembly, but blocks formation of infectious particles. In this report, we demonstrate that Gag proteins lacking the entire MHR accumulated in the cytoplasm of transfected COS-1 cells, as did the wild-type protein, but were processed in a defective manner at the cellular membrane resulting in impaired particle assembly. To further examine the role of the MHR in membrane association, membrane binding of unmyristylated recombinant Gag proteins with alterations in the MHR was investigated in vitro. The wild-type Gag precursor bound to acidic phospholipid vesicles highly efficiently, as determined by fluorescence spectroscopy or velocity sedimentation. In contrast, deletion of the entire MHR reduced membrane affinity an average of approximately 3-fold or greater. Mutation of the invariant Gln287 residue disrupted membrane affinity approximately 6-fold relative to the wild-type, which was similar to the level of inhibition obtained by deletion of a membrane-binding signal previously identified in the matrix domain of the Gag precursor. Mutation of the invariant Arg299 residue reduced the affinity to a lesser extent. The results indicate that correct membrane binding is determined not only by signals in the MA domain of the precursor but also by sequences in the CA domain of Gag. We speculate that defects in the highly conserved MHR affect a Gag conformation that is required for productive interactions at the membrane assembly site.