Cyclophilins are a family of proteins that bind the immunosuppressant cyclosporin A, possess peptidyl-prolyl cis-trans isomerase activity, and assist in the folding of proteins. Human cyclophilins A and B are host cell proteins that bind specifically to the HIV-1 Gag polyprotein p55gag in vitro. Here we report that viral particles formed by p55gag, in contrast to particles formed by the Gag polyproteins of other retroviruses, contain significant amounts of cyclophilin A. Sequences in the capsid domain of p55gag are both required and sufficient for the virion-association of cyclophilin A. The association of cyclophilin A with HIV-1 virions was inhibited in a dose-dependent manner by cyclosporin A as well as by SDZ NIM811 ([Melle-4]cyclosporin), a non-immunosuppressive analogue of cyclosporin A. Drug-induced reductions in virion-associated cyclophilin A levels were accompanied by reductions in virion infectivity, indicating that the association is functionally relevant. Moreover, SDZ NIM811 inhibited the replication of HIV-1 but was inactive against SIVMAC, a primate immunodeficiency virus closely related to HIV-1, which does not incorporate cyclophilin A.

Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.

Communicated by W.Schaffner enhancer motifs analyzed are involved in positive rather than negative control of transcription.

The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, 2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. 1 plays a critical role during the viral life cycle by mediating the attachment of virions to target cells and the fusion of viral and cellular membranes (1). Incorporation of Env, therefore, is essential for the formation of infectious viral particles. The cytosolic domain of the Env transmembrane subunit gp41 is the portion of the Env protein complex that is most likely to interact with the internal structural proteins of the virus (2-5). Although the cytosolic domain of Env is absolutely required for viral dissemination in vivo it seems to be dispensable for envelope incorporation into virions and, consequently, for viral replication in vitro (6). How newly assembled virions specifically acquire Env remains therefore largely unknown.An intriguing characteristic of the Env proteins of HIV-1 and simian immunodeficiency virus is that they undergo rapid endocytosis after their transport to the cell surface (7,8). As a consequence, the internal structural proteins of these viruses need to compete with the internalization machinery of the cell in order to acquire Env (9).2 Although the functional significance of this phenomenon is not understood, it is clear that Env behaves like other plasma membrane proteins that are rapidly internalized from the cell surface. Rapid internalization involves recruitment of plasma membrane proteins to clathrincoated pits, a process that is mediated by interaction of endocytic signals found in the cytosolic domains of the proteins with the clathrin-associated adaptor complex AP-2 (10 -12). The AP-2 complex consists of two large chains (␣ and ␤2), a medium chain (2), and a small chain (2). A direct interaction between 2 and tyrosine-based sorting signals from the cytosolic domains of several cellular integral membrane proteins has been recently demonstrated (13)(14)(15).To assess the diverse functions of the cytosolic domain of Env including its role in internalization from the plasma membrane, we are analyzing its interaction with cellular and viral proteins. Anti-Env antibodies allowed us to co-immunoprecipitate Env with the AP-2 complex from HIV-1-infected lymphocytes, demonstrating that these proteins associate in vivo. Using GST-Env tail fusion proteins, we then identified the 2 chain of AP-2 as a protein that interacts with the cytosolic domain of Env. Binding of 2 to the cytosolic domain of Env was dependent on the presence and the context of a tyrosinebased sorting motif that is crucial for Env internalization (7), but it was also influenced by a glycine residue that had not previously been identified to be important for efficient endosomal sort...

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gpl20 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gpl20. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gpl20 mutants by the six Fabs was studied. The patterns of sensitivity to particular gpl20 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals

HIV‐1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV‐1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live‐cell imaging techniques, by immunocytochemistry and by immuno‐electron microscopy that HIV‐1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage‐derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV‐1 morphogenesis is thoroughly rooted in the endosomal system.

The envelope glycoprotein (Env) is an essential component of retroviruses because it mediates the selective attachment of virus to its target cell (19). Env is not necessary for the formation and the release of retroviral particles, but Env of lentiviruses, like the glycoproteins of other enveloped RNA viruses, has been implicated in the spatial restriction of virus production (2, 5, 51). In addition, Env contributes to controlling the rate with which virions exit the host cell (44, 50). Env's position in the viral replication cycle is thus pivotal not only because it controls viral entry but also because it regulates when and where exactly virus will be released during the late phase of the viral life cycle.Polarization of lentiviral release is observed not only in epithelial cells but also in lymphocytes and probably in neurons (8,24,41,55). Such a directed release of virus may be particularly important for an efficient spread of virus in the crowded environment of lymph nodes, where infection is propagated locally, but polarized secretion is probably also important during systemic dissemination, when the virus crosses tissue barriers.How Env targets virus release to certain areas of the cell surface is not known. However, viral exit at distinct sites coincides with Env localization in these areas. It seems likely, therefore, that Env accumulation at distinct sites is a prerequisite for the polarized release of infectious particles. Consequently, signals which direct Env from the trans-Golgi network (TGN) to these sites are crucial for directional virus secretion (see, e.g., references 8 and 31).To elucidate how Env targets virus release to particular areas of the cell surface, we analyzed its trafficking during the late phase of the viral replication cycle. As part of these investigations we characterized the interaction of Env's cytosolic domain (EnvCD) with proteins of the cellular sorting machinery. The targeting of membrane proteins from the Golgi apparatus to the plasma membrane or to compartments of the endosomal system is mediated by specialized clathrin-coated vesicles (CCVs) (22,26,32,35,45). Endocytosis of membrane proteins gives rise to the formation of similar transport vesicles at the plasma membrane. Selection of the membrane proteins into CCVs largely depends on the interaction of signals in the cytosolic domains of these proteins with specific adapter proteins (APs). These APs are part of the vesicle coats forming at the cytosolic leaflet of the lipid bilayer. The existence of four

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp4l transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp4l glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp4l and the gpl20 exterior glycoprotein. Amino acid changes in other parts of the gp4l glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp4l human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp4l ectodomain. Human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS, which is characterized by the depletion of CD4-positive lymphocytes (6, 26, 33). HIV-1 exhibits a tropism for CD4-bearing cells that is mediated by a specific interaction between the viral envelope glycoproteins and the CD4 glycoprotein (18, 44, 53, 55). The HIV-1 envelope glycoproteins are synthesized as a 160-kDa precursor (gp160) that is cleaved into the exterior envelope glycoprotein (gpl20), which binds to the CD4 molecule, and a transmembrane glycoprotein (gp4l) (1, 70, 82). The HIV-1 gpl20 and gp41 glycoproteins remain associated through noncovalent interactions (38, 46, 56). Following CD4 binding, the envelope glycoproteins mediate a pH-independent process that results in the fusion of the viral and target cell membranes and that is necessary for virus entry (37, 77). HIV-1 envelope glycoproteins expressed on the surface of infected cells initiate receptor-binding and membrane fusion events with adjacent CD4-positive cells, resulting in the formation of multinucleated syncytia (52, 75). Structural and mutagenic studies have provided some insight into the molecular events underlying HIV-1 entry and syncytium formation. The CD4-binding region of gpl20 consists of a conformation-dependent structure with elements derived from discontinuous sequences in the third and fourth conserved regions of the glycoprotein (16, 17, 46, 50, 62). The gpl20 glycoprotein binds to the CD4 region bearing similarity to the second complementarity-determining (CDR2) region of immunoglobulins (3, 4, 7, 11, 12, 14, 15, 42, 49, 57, 66). The CDR2-like region of CD4, located in the aminoterminal domain of the glycopro...