Maturation-induced conformational changes of HIV-1 capsid protein and identification of two high affinity sites for cyclophilins in the C-terminal domain.
(1/922)
Viral incorporation of cyclophilin A (CyPA) during the assembly of human immunodeficiency virus type-1 (HIV-1) is crucial for efficient viral replication. CyPA binds to the previously identified Gly-Pro90 site of the capsid protein p24, but its role remained unclear. Here we report two new interaction sites between cyclophilins and p24. Both are located in the C-terminal domain of p24 around Gly-Pro157 and Gly-Pro224. Peptides corresponding to these regions showed higher affinities (Kd approximately 0.3 microM) for both CyPA and cyclophilin B than the best peptide derived from the Gly-Pro90 site ( approximately 8 microM) and thus revealed new sequence motifs flanking Gly-Pro that are important for tight interaction of peptide ligands with cyclophilins. Between CyPA and an immature (unprocessed) form of p24, a Kd of approximately 8 microM was measured, which corresponded with the Kd of the best of the Gly-Pro90 peptides, indicating an association via this site. Processing of immature p24 by the viral protease, yielding mature p24, elicited a conformational change in its C-terminal domain that was signaled by the covalently attached fluorescence label acrylodan. Consequently, CyPA and cyclophilin B bound with much higher affinities ( approximately 0.6 and 0.25 microM) to the new, i.e. maturation-generated sites. Since this domain is essential for p24 oligomerization and capsid cone formation, CyPA bound to the new sites might impair the regularity of the capsid cone and thus facilitate in vivo core disassembly after host infection. (+info)
Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab.
(2/922)
The crystal structure of an intact molecule of HIV-1 capsid protein (p24) in complex with a monoclonal antibody fragment recognizing an epitope on the C-terminal domain has been determined at 3 A resolution. The helical N- and C-terminal domains of p24 are linked by an extended peptide forming a flexibly linked dumb-bell-shaped molecule 75 A in overall length. The p24 construct used is a variant with an N-terminal extension that mimics to some extent the Gag context of p24. We observed a novel head-to-tail dimer of p24 molecules which occurs through the formation of a substantial intermolecular interface between the N- and C-terminal domains. Comparison with previously observed p24 dimers shows that the same residues and secondary structural elements can partake in different interfaces revealing a remarkable stickiness and plasticity of the p24 molecule, properties which, combined with the inter-domain flexibility, are presumably important in the assembly and maturation of viral particles. Previous mutagenesis studies designed to test specific N-N and C-C homodimer interfaces do not discriminate fully against the possibility of the observed N-C interface. (+info)
Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.
(3/922)
It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion. (+info)
Evolution and biological characterization of human immunodeficiency virus type 1 subtype E gp120 V3 sequences following horizontal and vertical virus transmission in a single family.
(4/922)
It has been suggested that immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the third variable (V3) loop of the gp120 of human immunodeficiency virus type 1 (HIV-1). Here we present evidence, on the basis of sequencing 147 independently cloned env C2/V3 segments from a single family (father, mother, and their child), that the intensity of positive selection is related to the V3 lineage. Phylogenetic analysis and amino acid comparison of env C2/V3 and gag p17/24 regions indicated that a single HIV-1 subtype E source had infected the family. The analyses of unique env C2/V3 clones revealed that two V3 lineage groups had evolved in the parents. Group 1 was maintained with low variation in all three family members regardless of the clinical state or the length of infection, whereas group 2 was only present in symptomatic individuals and was more positively charged and diverse than group 1. Only virus isolates carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells, a transformed CD4(+)-T-cell line. A statistically significant excess of nonsynonymous substitutions versus synonymous substitutions was demonstrated only for the group 2 V3 region. The data suggest that HIV-1 variants, possessing the more homogeneous group 1 V3 element and exhibiting the non-syncytium-inducing phenotype, persist in infected individuals independent of clinical status and appear to be more resistant to positive selection pressure. (+info)
Rate and severity of HIV-associated dementia (HAD): correlations with Gp41 and iNOS.
(5/922)
BACKGROUND: Fifteen to thirty percent of AIDS patients develop some type of neurologic disorder during the course of their illness and the vast majority of these neurologic disorders will be HIV-associated dementia (HAD). These patients can exhibit varying degrees of severity and rates of progression of HAD. Neuropathologic variables that are associated with the rate of progression of HAD are not known. MATERIALS AND METHODS: Tissue was collected at autopsy from the Johns Hopkins University HIV Neurology Program. Seventy-one AIDS patients of this prospectively characterized population were followed until death to obtain information on dementia severity and the rate of neurological progression. Immunoblot analysis of immunological nitric oxide synthase (iNOS), HAM56, gp41, p24, gp120, and beta-tubulin was performed and the levels of iNOS, HAM56, gp41, and p24 were normalized to beta-tubulin and analyzed for significance by means of the Kruskal-Wallis test for multiple groups. RESULTS: We have identified unique groups within this spectrum and designated them slow, moderate, and rapid progressors. Slow and moderate progressors' neurological progression occurs over a course of months to years, whereas the rapid progressors' disease shows rapid increases in severity over weeks to months. In the present study we demonstrate that the severity and rate of progression of HAD correlates significantly with levels of the HIV-1 coat protein, gp41, iNOS, and HAM56, a marker of microglial/macrophage activation. CONCLUSION: The severity and rate of progression of HAD correlates with indices of immune activation as well as levels of iNOS and gp41. There appears to be a threshold effect in which high levels of gp41, iNOS, and immune activation are particularly associated with severe (Memorial Sloan-Kettering score 3 to 4) and rapidly progressive HAD. (+info)
Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments.
(6/922)
Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively). (+info)
Comparison of the frequencies and levels of human immunodeficiency virus type 1 markers in specimens from chronically infected human T-lymphocyte cultures and from patients.
(7/922)
Together with CD4(+)-cell counts as an indicator of immune function, the use of human immunodeficiency virus type 1 (HIV-1) RNA levels as a direct marker of viral load has gained widespread attention for evaluation of patient clinical status. Results obtained with other HIV-1 markers for this purpose are often inconsistent. This study examined the relationship between various HIV-1 markers by using clinical specimens (plasma) from HIV-1-infected individuals at different stages of disease progression and supernatant fluid from four human T-lymphocyte cell lines chronically infected with HIV-1. Cell culture specimens were collected periodically over 7 days and were tested for HIV-1 RNA levels with a nucleic acid amplification assay, for p24 with an enzyme-linked immunosorbent assay, and for reverse transcriptase activity by isotope uptake. An increase in the level of each marker was observed over the 7-day period with each of the four HIV-1 strains tested (LAV1, HTLV-IIIB, MN, and ARV2); with these specimens, the frequency of detection for each marker was 100%. In the clinical specimens, HIV-1 RNA was detected more often (143 of 183 specimens [78%]) than was p24 (87 of 183 [48%]); little correlation between the levels of the two markers was seen. In these clinical specimens evaluated, CD4(+)-cell counts were better correlated with the frequency and levels of HIV-1 RNA than with p24. In specimens (n = 38) collected serially from six HIV-1-infected subjects, HIV-1 RNA was detected more often (33 of 38 [85%]) than p24 (23 of 38 [59%]). When reported by the assays used, the levels of both HIV-1 markers fluctuated over time for each of the subjects. Although the markers correlated in the in vitro systems studied, the observed differences in the correlation of levels and frequencies of HIV-1 markers in vivo indicate that p24 has less clinical utility than does viral load testing when used in conjunction with CD4(+)-cell counts as a measure of immune system functioning. (+info)
Decreased CD8 cell-mediated viral suppression and other immunologic characteristics of women who transmit human immunodeficiency virus to their infants.
(8/922)
CD8 T cell function, lymphocyte surface phenotype, serum markers of immunologic activation, and viral burden were assessed in 75 human immunodeficiency virus (HIV)-infected pregnant women, including 9 who transmitted infection to their infants. Serial studies during and after pregnancy showed no significant differences in levels of cell-surface or serum activation molecules in transmitting compared to nontransmitting mothers, with the exception of a postpartum increase in tumor necrosis factor alpha in transmitting women. The transmitting women had a median plasma viral load of 65,516 RNA copies/mL at delivery versus 5139 in nontransmitting women. During the third trimester, the CD8 cells of 81% of the nontransmitting and 44% of the transmitting mothers suppressed HIV production in vitro by >50%. Women with <50% suppression had a 3.4 times greater risk of transmitting HIV to their infants. CD8 suppression and viral load were interrelated, but when either CD4 percent or AZT use was controlled for, suppression was still significant. (+info)