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Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV‐1 Nef non‐covalently associates with actin forming a high‐molecular‐mass complex of 150–300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N‐terminal myristoylation of Nef, whereas the SH3‐binding proline motif of Nef was not involved. Despite being myristoylated, HIV‐2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell‐fractionation experiments revealed that HIV‐ 1Nef was virtually exclusively localized in the cytoskeletal (detergent‐insoluble) fraction whereas HIV‐ 2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV‐1‐in‐fected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV‐infected cells. This novel interaction of HIV‐1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV‐1 and HIV‐2.

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RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines. (Journal of Biomolecular Screening 2009:151-160)