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1.
Open AIDS J ; 3: 4-7, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-19554213

ABSTRACT

The presence of antigen-specific cellular immune responses may be an indicator of long-term asymptomatic HIV-1-disease. The detection of cellular immune responses to infection with different subtypes of HIV-1 may be hampered by genetic differences of immunodominant antigens such as the capsid protein CAp24. In Nouna, Burkina Faso, HIV-1 circulating recombinant forms CRF02_AG and CRF06_cpx are the 2 major strains detectable in HIV-1-infected individuals, while subtype B strains prevail in Europe and North America. Amino acid sequences of CAp24 were assessed in blood samples from 10 HIV-1-infected patients in Nouna, Burkina Faso. Production of interferon-gamma (IFN-gamma) in peripheral blood CD4(+) lymphocytes in response to recombinant HIV-1 proteins derived from clade B (including CAp24(NL4-3)) was measured using a modified flow-cytometry-based whole blood short term activation assay (FASTimmune, BDBiosciences). IFN-gamma production following stimulation with a whole length CAp24 protein derived from clade B (CAp24(NL4-3)) was additionally quantified in comparison to a CAp24 protein derived from CRF02_AG (CAp24(BD6-15)) in 16 HIV-1-infected patients in Heidelberg, Germany. Amino acid sequence identity of CAp24 obtained from patients in Nouna ranged between 86 and 89% when compared to the clade B CAp24(NL4-3) consensus sequence, between 90 and 95% when compared to the circulating recombinant form CRF06_CPX consensus sequence, and between 92 and 96% when compared to the CAp24(BD6-15) consensus sequence. Significant numbers of HIV-1-specific CD4(+) lymphocytes producing IFN-gamma were detected in 4 of 10 HIV-1-infected patients. In 7 of 16 patients in Heidelberg, recombinant CAp24(BD6-15) stimulated IFN-gamma-production in CD4(+) lymphocytes to a similar extent as the clade B-derived CAp24(NL4-3). Thus, antigen-specific CD4(+) lymphocytes from both West African and European patients infected with different strains of HIV-1 show relevant cross-clade recognition of HIV-1 CAp24 in a flow-cytometry-based whole blood short term activation assay.

2.
J Biol Chem ; 283(46): 32024-33, 2008 Nov 14.
Article in English | MEDLINE | ID: mdl-18772135

ABSTRACT

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.


Subject(s)
Capsid Proteins/chemistry , Capsid Proteins/metabolism , Capsid/drug effects , Capsid/metabolism , HIV-1/chemistry , HIV-1/metabolism , Virus Assembly/drug effects , Amino Acid Sequence , Binding Sites , Capsid Proteins/genetics , Capsid Proteins/ultrastructure , Cell Line , Conserved Sequence , Crystallography, X-Ray , HIV-1/drug effects , HIV-1/ultrastructure , Humans , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Peptides/pharmacology , Protein Structure, Quaternary , Sequence Alignment
3.
Antiviral Res ; 78(3): 275-7, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18329737

ABSTRACT

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.


Subject(s)
Antibodies, Monoclonal/pharmacology , Drug Resistance, Viral/genetics , Genetic Variation , HIV Protease/drug effects , HIV-1/enzymology , Immunoglobulin Fragments/pharmacology , Recombinant Proteins/pharmacology , Antibodies, Monoclonal/immunology , Antiretroviral Therapy, Highly Active , Dimerization , HIV Infections/drug therapy , HIV Infections/virology , HIV Protease/genetics , HIV Protease/immunology , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , HIV-1/genetics , Humans , Immunoglobulin Fragments/immunology , Models, Molecular , Mutation , Recombinant Proteins/immunology
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