Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a P-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 Angstrom N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba(67,95)]HIVPR and [Lys(7),Ile(33),Aba(67,95)]- HIVPR used in this work were shown to have very similar crystal structures.

Influence of HIV-1 coinfection on effective management of abnormal vaginal discharge

Background: Reports on microbiologic cure rates following syndromic management (SM) of women with nonulcerative sexually transmitted infections (STIs) are limited. Goal. The goal of the study was to determine the effectiveness of the drugs used in SM of nonulcerative STIs and bacterial vaginosis in women and to compare the response among those with and without HIV-1 coinfection. Study Design: This was a cohort study of women with nonulcerative STIs who were treated according to local SM protocols. Results: Of 692 women recruited, 415 (80%) returned 8 to 10 days later, and 290 (70%) consented to a second examination, in which specimens were obtained. Clinical cure was reported by 67%, and microbiologic cure ranged from 80% to 89% for the three discharge-causing STIs and was independent of HIV-1 status. Only 38% of those with bacterial vaginosis were cured, and HIV-1-infected women were less likely to be cured (28% versus 52%; P < 0.001). Conclusions: Clinical and microbiologic response to SM of the nonulcerative STIs was not affected by HIV-1 coinfection, but cure rates for bacterial vaginosis were reduced.

Association between HIV-1 infection, the etiology of genital ulcer disease, and response to syndromic management

Background: Reports on the effect of HIV-1 infection on healing rates of ulcers are conflicting. Goal: The goal was to determine the etiology and response to treatment of genital ulcer disease (GUD) in relation to HIV-1 infection. Study Design: This was a cohort study of patients with GUD treated with local syndromic management protocols. Results: Among the 587 recruited, the prevalences of infections due to HSV, Treponema pallidum, Chlamydia trachomatis (lymphogranuloma venereum [LGV]), Haemophilus ducreyi, Calymmatobacterium granulomatis, and HIV-1 were 48%, 14%, 11%, 10%, 1%, and 75%, respectively. The prevalence of T pallidum was higher among men (P = 0.03), and an association was seen among HIV-1-seronegatives on univariate and multivariate analyses (P < 0.001; P = 0.01). The prevalence of C trachomatis (LGV) was higher among females (P = 0.004), and an association was seen among HIV-1-seropositives on univariate analysis (P = 0.04). At follow-up, 40/407 (10%) showed a decreased healing tendency, not associated with ulcer etiology or HIV-1 seropositivity. Conclusion: Response to syndromic management of GUD was acceptable and not associated with HIV-1 coinfection.

Synthetic studies of the HIV-1 protease inhibitive didemnaketals: Stereocontrolled synthetic approach to the key mother spiroketals

[GRAPHICS] The stereocontrolled synthesis of (2S,4R,6R,8S,10S,1'R,1"R)-2(acetylhydroxymethyl)-4, 10-dimethyl-8(isopropenylhydroxymethyl)-1, 7-dioxaspiro[5,5]-undecane (4a) and its C1"-epimer (4b), the key mother spiroketals of the HIV-1 protease inhibitive didemnaketals from the ascidian Didemnum sp., has been carried out through multisteps from the natural (R)-(+)-pulegone, which involved the diastereoselective construction of four chiral carbon centers(C-2, C-6, C-8, and C-1') by intramolecular chiral induce.

Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class IKO mice

HLA-A*0201 transgenic, H-2D(b)/mouse beta2-microglobulin double-knockout mice were used to compare and optimize the immunogenic potential of 17HIV 1-derived, HLA-A0201-restricted epitopic peptides. A tyrosine substitution in position 1 of the epitopic peptides, which increases both their affinity for and their HLA-A0201 molecule stabilizing capacity, was introduced in a significant proportion, having verified that such modifications enhance their immunogenicity in respect of their natural antigenicity. Based on these results, a 13-polyepitope construct was inserted in the pre-S2 segment of the hepatitis B middle glycoprotein and used for DNA immunization. Long-lasting CTL responses against most of the inserted epitopes could be elicited simultaneously in a single animal with cross-recognition in several cases of their most common natural variants.

Mechanistic aspects of HIV-1 reverse transcription initiation

During reverse transcription, the positive-strand HIV-1 RNA genome is converted into a double-stranded DNA copy which can be permanently integrated into the host cell genome. Recent analyses show that HIV-1 reverse transcription is a highly regulated process. The initiation reaction can be distinguished from a subsequent elongation reaction carried out by a reverse transcription complex composed of (at least) heterodimeric reverse transcriptase, cellular tRNA(lys3) and HIV-1 genomic RNA sequences. In addition, viral factors including Tat, Nef, Vif, Vpr, IN and NCp7, cellular proteins, and TAR RNA and other RNA stem-loop structures appear to influence this complex and contribute to the efficiency of the initiation reaction. As viral resistance to many antiretroviral compounds is a continuing problem, understanding the ways in which these factors influence the reverse transcription complex will likely lead to novel antiretroviral strategies. Copyright (C) 2001 John Wiley Sons, Ltd.

The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions

Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.