In contrast to HIV-1, lentiviruses of the HIV-2/SIVmac/SIVsm lineage encode an additional accessory protein, Vpx. Vpx, like the related HIV-1 accessory protein Vpr, is packaged into virions through a virus-specific interaction with the p6 domain of Gag. Its presence in the virion suggests that Vpx acts post-entry. Although the molecular function of Vpx within the host cell is still poorly understood, recent evidence has suggested that Vpx counteracts a yet unidentified restriction factor in macrophages and dendritic cells. In this study, Vpx was found to facilitate infection of monocytederived macrophages (MDM) and monocyte-derived dendritic cells (MDDC), key cellular targets for the virus in vivo. Analysis of reverse transcription products by real-time PCR showed that Vpx overcomes a block to reverse transcription. In a mutagenesis study, amino acid residues near the amino terminus of Vpx were found to be required for biological function. These mutants retained the ability to bind DCAF1, suggesting that this domain serves as a binding site for the restriction factor. Although HIV-1 does not encode Vpx, the protein can be provided to MDM and MDDC in virus-like particles, dramatically enhancing their susceptibility to HIV-1. A genetically modified form of HIV-1 was developed that would package the protein into virions. To do this, the minimal Vpx packaging motif of SIVmac₂₃₉ was identified and then introduced into the molecular HIV-1 clone pNL4-3. The chimeric virus packaged SIVmac Vpx and was substantially more infectious on MDDC and MDM than the wild-type virus. The virus was further modified by introducing vpx in place of nef. The resulting virus produced Vpx and replicated more efficiently in MDDC and MDM. The virus also induced a high level of IFNβ in MDDC. In a coculture system, Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo Vpx may facilitate dendritic cell to T cell virus transmission. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.