Viral protein X (vpx) is a virion associated HIV-2/SIV accessory protein that enhances viral infectivity and replication in natural target cells. To investigate whether other viral components affect its biosynthesis, subcellular localization, and virion incorporation, we expressed HIV-2 vpx in a mammalian cell system and examined its transport and packaging requirements using an in trans complementation assay. The complete vpx coding region of HIV-2ST was placed under the control of a high-efficiency promoter system (SRα) which contained both an SV40 promoter/enhancer region and R/U5 elements of the HTLV-1 LTR. Following transfection of Cos-1 cells, this construct (pSRα-vpx) facilitated high level expression of vpx, as demonstrated by Western blot analysis of transfected cell lysates. Moreover, indirect immunofluorescence analysis revealed an intense vpx staining pattern distributed evenly throughout the cytoplasm of transfected cells. This distribution differed markedly from cells expressing wild-type HIV-2 in which vpx localized to the inner surface of the plasma membrane. To determine whether other HIV components were required for this surface localization, we expressed vpx in the context of replication competent HIV-1 and HIV-2 proviruses. Following cotransfection with a vpx-deficient HIV-2 provirus (pXM7), eukaryotically expressed vpx targeted to the plasma membrane and colocalized with HIV-2 p27 gag in a pattern indistinguishable from wild-type HIV-2. Moreover, progeny virions from cotransfected Cos-1 cells contained wild-type amounts of vpx protein, demonstrating that vpx could be efficiently packaged in trans. Under the same experimental conditions, cotransfection of vpx with wild-type HIV-1 (pHXB2) and with vpr-deficient HIV-1 (pR2) failed to result in detectable cell surface targeting or virion incorporation of vpx despite its high level cellular expression. These results demonstrate that efficient intracellular transport and packaging of vpx require interaction with other type-specific virus components. © 1993 Academic Press, Inc.