Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis.

The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N.

Engineering a rigid protein tunnel for biomolecular detection.

One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore.

Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.

Background: Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution.

Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA.

The mature nucleocapsid protein of HIV-1, NCp7, and the NC domains in gag precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion.

Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 Vif and inhibitors of Vif-APOBEC3G binding.

The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown.

Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation.

HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box.

Specificity of neomycin analogues bound to the packaging region of human immunodeficiency virus type 1 RNA.

The packaging region of HIV-1 RNA contains a number of structural features which are important in the life cycle of the virus, making this segment of RNA a potential target for new types of AIDS-directed drugs. We studied the binding of three neomycin analogues (neo-guanidino, neo-acridine, and neo-neo) to a 171-mer RNA molecule from the packaging region of HIV-1 using quantitative footprinting and circular dichroism. Neo-guanidino produced footprinting patterns and effects on the CD similar to those observed for neomycin and paromomycin, indicating that all three compounds bind to the same regions of the 171-mer.

Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway.

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious.

Footprinting and circular dichroism studies on paromomycin binding to the packaging region of human immunodeficiency virus type-1.

We have studied the interaction of the aminoglycoside drug, paromomycin, with a 171-mer from the packaging region of HIV-1 (psi-RNA), using quantitative footprinting and circular dichroism spectroscopy. The footprinting autoradiographic data were obtained by cutting end-labeled RNA with RNase I or RNase T1 in the presence of varying paromomycin concentrations. Scanning the autoradiograms produced footprinting plots showing cleavage intensities for specific sites on the psi-RNA as functions of drug concentration.

Footprinting, circular dichroism and UV melting studies on neomycin B binding to the packaging region of human immunodeficiency virus type-1 RNA.

We have studied the binding of neomycin to a 171mer RNA (psi-RNA) from the packaging region of the LAI strain of human immunodeficiency virus type-1, HIV-1 (LAI). The RNase I footprinting studies reveal that the primary binding site for the drug is in stem-loop 1, which contains the dimer initiation site of HIV-1. Loading this site with neomycin causes a structural change in the RNA, allowing nucleotides in the neighboring stem-loop 2 to participate in the drug site.