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Infection by Human Immunodeficiency Virus is difficult to treat thanks to its persistent viral reservoir and to its high rate of mutation that allows appearance of resistance to the available treatment. An approach to discover drugable targets is the identification of cellular partner proteins interacting with the virus during its life cycle. We used RNAi technology to identify new HIV partners in the host cytoskeleton since it has been shown that cytoskeleton components and the irregulators have a role during several steps of HIV life cycle. By transducingseveral Tcell lines such as Jurkat CD4 CCR5, Jurkat E6-1 and SupT1 with lentiviral vectors expressings hRNA sequences, we silenced different target genes, members of pathways involved inactinrearrangement. By infection with HIV-NL4.3-eGFP reporter virus we evaluated HIV replication ratesin transducedcells. Surprisingly, the infection rate affected by the specific knock-down was dependent on the cell line used. lndeed, ashRNAtransduced in one cell line affected infection differently to what it did in another. Moreover, we observed that transduction on itself with a control vector expressinga scrambled shRNA sequence affected HIV infection rate in some but not all cell lines. Therefore, to obtain relevant results in screening co-factors for HIV infection, we turned toprimary cells, the naturaltargets of the virus in vivo. We optimized combinedlentiviral transduction and HIV infection in cultured peripheral blood CD4+ lymphocytes. In this setting, transduction with scrambled shRNA expressing lentivirus did not affect HIV replication, providing us a platform to assay gene-knockdown likely to generate the most relevant information for natural HIV infection invivo.

RNAi is a sequence-specific gene silencing mechanism induced by double-stranded RNA and a promising tool for the development of a durable gene therapy against HIV-1. Such RNAi attack usually focuses on targets within the HIV-1 RNA genome. Despite potent inhibition, we previously observed the rapid selection of RNAi-resistant HIV-1 variants with a mutated target sequence. A number of strategies were tested to prevent viral escape. Similar to the success of combination therapy with multiple anti-retroviral drugs, one can target HIV-1 with multiple short hairpin RNAs (shRNAs). Alternatively, one can design second-generation shRNAs to target favorite RNAi-resistance mutations in order to block specific viral escape routes. One could also combine RNAi with regular antiviral drug regimens. Another strategy involves the use of RNAi against cellular proteins that are involved in HIV-1 replication. The idea is that viral escape is more difficult in this setting. Many cellular co-factors have been described in literature, e.g. based on large RNAi-screens in cell lines. We have shown that long-term inhibition of HIV-1 replication is possible with RNAi against cellular co-factors. To identify relevant co-factors that are not essential for cellular function, primary cells are to be preferred over T cell lines. We propose that RNAi against both viral and cellular targets could be used as a new therapeutic approach in the combat against HIV-1 infection.