Background: The integration of HIV-1 DNA into cellular chromatin is required for high levels of viral gene expression and for the production of new virions. However, the majority of HIV-1 DNA remains unintegrated and is generally considered a replicative dead-end. A limited amount of early gene expression from unintegrated DNA has been reported, but viral replication does not proceed further in cells which contain only unintegrated DNA. Multiple infection of cells is common, and cells that are productively infected with an integrated provirus frequently also contain unintegrated HIV-1 DNA. Here we examine the influence of an integrated provirus on unintegrated HIV-1 DNA (uDNA).

Unlike activated T cells, quiescent CD4Together, these data demonstrate that the early stages of the HIV life cycle are inefficient in these poststimulated cells and that efficient replication cannot be induced by subsequent activation. Unlike their stimulated counterparts, quiescent CD4ϩ T cells have demonstrated resistance to human immunodeficiency virus (HIV) infection. Our earlier studies showed that quiescent CD4 ϩ T cells, when infected with HIV, were unable to support productive viral replication, characterized by incompletely reverse transcribed viral DNA (47, 48). However, the permissiveness of other quiescent cell types, such as macrophages (12,28,35,36,39), raised further questions regarding the exact nature of the block of HIV infection in quiescent CD4 ϩ T cells. Cells in the G 0/1a stage of the cell cycle are truly quiescent, while cells in G 1b are characterized by high levels of RNA synthesis in the absence of DNA synthesis (10,45). Further studies demonstrated that T cells at the G 0/1a stage of the cell cycle are truly resistant to viral infection, whereas cells in the G 1b phase are permissive (21). Thus, T cells need not be actively dividing to support a productive HIV infection, a phenomenon also seen in macrophages (36). Treatment of G 0/1a cells with nucleotides resulted in completion of reverse transcription (RT) but was insufficient to rescue productive viral infection (22). Therefore, the block in HIV replication was not merely a lack of raw materials but possibly of crucial cellular factors or the presence of inhibitors. This hypothesis was supported by subsequent studies demonstrating that reverse transcription of viral vectors was slow in quiescent cells, requiring 2 to 3 days, and that the half-life of the full-length viral genome was approximately 1 day (31, 50). Alternative methods of infection, such as spinoculation, while increasing the amount of viral entry, do not overcome the block in HIV infection (30,43,44). A number of proteins have been suggested to inhibit HIV-1 replication in CD4ϩ T cells, such as Murr1 and APOBEC3G. Decreased Murr1 expression rendered quiescent CD4 ϩ T cells permissive to HIV infection (13). APOBEC3G, a cytidine deaminase, has been shown to cause high levels of hypermutation of the HIV genome (15,24,49). The effects of APOBEC3G are countered by a viral protein, Vif, which has been shown to degrade APOBEC3G and prevent it from being packaged in the virion (9,20,26,27,37,42,46). Recently, Chiu et al. showed that APOBEC3G exists in an active enzymatic form at the G 0/1a stage of the cell cycle, which inhibits HIV infection at the level of reverse transcription, although not by cytidine deaminase activity (5).To further characterize the block of HIV infection in quiescent CD4ϩ T cells, we infected highly purified cells with the CXCR4-tropic HIV-1 NL4-3 and stimulated (17, 19) these cells before (prestimulated) or 2 h after (poststimulated) infection. Quiescent cells displayed slightly lower levels of viral entry than stimulated cells. We also analyzed infe...

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201–restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8 + T-cell population. Following tumor challenge, these transgenic CD8 + T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non–HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.