Nef Alleles from All Major HIV-1 Clades Activate Src-Family Kinases and Enhance HIV-1 Replication in an Inhibitor-Sensitive Manner

The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk

The groningen longitudinal glaucoma study III. The predictive value of frequency-doubling perimetry and GDx nerve fibre analyser test results for the development of glaucomatous visual field loss

Purpose To investigate whether frequency-doubling perimetry (FDT) and nerve fibre analyser (GDx) test results are able to predict glaucomatous visual field loss in glaucoma suspect patients. Methods A large cohort of glaucoma suspect patients (patients with ocular hypertension or a positive family history of glaucoma without visual field abnormalities at baseline) was followed prospectively for 4 years with SAP (HFA 30-2 SITA fast), FDT (C-20 full threshold), and GDx (version 2.010) in a clinical setting. After the follow-up period, baseline FDT and GDx test results of converters (glaucoma suspect patients who had converted to a reproducible abnormal SAP test result during follow-up) were compared to that of non-converters (suspects with normal SAP test results at the end of the follow-up) by calculating relative risks. Cutoff point for FDT was >1 depressed test point P29. Results Of 174 glaucoma suspect patients, 26 had developed reproducible glaucomatous visual field loss (conversion rate: 3.7% per year). Relative risk was 1.8 (95% confidence interval: 0.9-3.7; P = 0.10) for FDT and 2.7 (95% confidence interval: 1.2-6.3; P = 0.01) for GDx. Positive predictive value was 0.22 for both FDT and GDx; negative predictive value was 0.88 for FDT and 0.92 for GDx. Conclusions In a clinical setting, especially GDx may be helpful for identifying glaucoma suspect patients at risk of developing glaucomatous visual field loss as assessed by SAP. Eye (2009) 23, 1647-1652; doi: 10.1038/eye.2008.348; published online 14 November 200

Color Doppler imaging in glaucoma patients with asymmetric visual field loss

WOS: 000088656400016With color Doppler imaging, we attempted to determine whether glaucoma patients with asymmetric visual field losses had evidence of asymmetric blood flow velocities in the central retinal artery despite similar intraocular pressure (IOP) curves in both eyes. We found that eyes with more severe visual field damage had an increased local resistance to blood flow in the central retinal artery. Thus vascular factors might have important roles in the pathogenesis of primary open-angle glaucoma