Elucidating a key anti-HIV-1 and cancer-associated axis: the structure of CCL5 (Rantes) in complex with CCR5.

CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

A trimeric structural domain of the HIV-1 transmembrane glycoprotein.

Infection with HIV-1 is initiated by fusion of cellular and viral membranes. The gp41 subunit of the HIV-1 envelope plays a major role in this process, but the structure of gp41 is unknown. We have identified a stable, proteinase-resistant structure comprising two peptides, N-51 and C-43, derived from a recombinant protein fragment of the gp41 ectodomain. In isolation, N-51 is predominantly aggregated and C-43 is unfolded. When mixed, however, these peptides associate to form a stable, alpha-helical, discrete trimer of heterodimers. Proteolysis experiments indicate that the relative orientation of the N-51 and C-43 helices in the complex is antiparallel. We propose that N-51 forms an interior, parallel, homotrimeric, coiled-coil core, against which three C-43 helices pack in an antiparallel fashion. We suggest that this alpha-helical, trimeric complex is the core of the fusion-competent state of the HIV-1 envelope.

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.

Structural comparison of the external glycoproteins of human and simian immunodeficiency virus.

The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.

HLA-DR peptide inhibits HIV-induced syncytia.

The infectivity of the human immunodeficiency virus (HIV) is related to the structure of its envelope protein, gp160, which is responsible for viral entry. We considered the possibility that a structural homology between gp160 and major histocompatibility complex (MHC) molecules might be associated with the extraordinary affinity that gp120 has for its receptor, CD4. Amino acid sequence comparisons revealed five regions of structural similarity between the HLA-DR beta molecule and gp160. The DR2 beta synthetic peptides containing these regions were examined for their ability to block HIV-induced syncytia formation using a 51Cr release assay. The peptide beta 141-155 inhibited the formation of syncytia whereas the other four DR beta peptides with gp160 similarity did not. Our results indicate that this region in gp120, which is similar to an HLA-DR region, is crucial to T cell-gp120 interactions, and should be considered in the design of future vaccines.