The interaction between the first and last intron nucleotides in the second step of pre-mRNA splicing is independent of other conserved intron nucleotides.

Virtually all pre-mRNA introns begin with the sequence /GU and end with AG/ (where / indicates a border between an exon and an intron). We have previously shown that the G residues at the first and last positions of the yeast actin intron interact during the second step of splicing. In this work, we ask if other highly conserved intron nucleotides also take part in this /G-G/ interaction. Of special interest is the penultimate intron nucleotide (AG/), which is important for the second step of splicing and is in proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict interactions between sets of three conserved intron nucleotides. In addition, we used random mutagenesis and genetic selection to search for interactions between nucleotides in the pre-mRNA. We find no evidence for other interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.     

Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino-acid sequences by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.     

A quantitative basis for antiretroviral therapy for HIV-1 infection

Highly active antiretroviral therapy (HAART) has dramatically decreased mortality from HIV-1 infection and is a major achievement of modern medicine. However, there is no fundamental theory of HAART. Elegant models describe the dynamics of viral replication, but a metric for the antiviral activity of drug combinations relative to a target value needed for control of replication is lacking. Treatment guidelines are based on empirical results of clinical trials in which other factors such as regimen tolerability also affect outcome. Why only certain drug combinations control viral replication remains unclear. Here we quantify the intrinsic antiviral activity of antiretroviral drug combinations. We show that most single antiretroviral drugs show previously unappreciated complex nonlinear pharmacodynamics that determine their inhibitory potential at clinical concentrations. We demonstrate that neither of the major theories for drug combinations accurately predicts the combined effects of multiple antiretrovirals. However, the combined effects can be understood with a new approach that considers the degree of independence of drug effects. This analysis allows a direct comparison of the inhibitory potential of different drug combinations under clinical concentrations, reconciles the results of clinical trials, defines a target level of inhibition associated with treatment success and provides a rational basis for treatment simplification and optimization.     

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.