ABSTRACT
BACKGROUND: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. METHODS AND FINDINGS: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. CONCLUSION: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes.
Subject(s)
HIV-1/genetics , HIV-1/metabolism , Monocytes/virology , Viral Regulatory and Accessory Proteins/metabolism , Virus Replication , Animals , Blotting, Western , Cercocebus , Genetic Vectors , HeLa Cells , Humans , Models, Genetic , Monocytes/metabolism , Simian Immunodeficiency Virus/metabolism , vpr Gene Products, Human Immunodeficiency Virus/genetics , vpr Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.
Subject(s)
Gene Expression Regulation , Nucleoside Deaminases/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , APOBEC-3G Deaminase , Base Sequence , Binding Sites , Cell Line , Cytidine Deaminase , Humans , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA Interference , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/genetics , Sp3 Transcription Factor/antagonists & inhibitors , Sp3 Transcription Factor/genetics , T-Lymphocytes/metabolism , Transcription Initiation Site , Transcription, GeneticABSTRACT
Although lentiviruses like HIV-1 are able to infect non-dividing cells, particular resting cells such as non-stimulated primary peripheral blood mononuclear cells (PBMC) are resistant to infection. In contrast to other lentiviruses, SIVsmmPBj can replicate in non-stimulated PBMC. Moreover, SIVsmmPBj-derived, but not HIV-1-derived, replication-incompetent vectors enable gene transfer into G(0)-arrested human cell lines and primary human monocytes. Here, we demonstrate that transduction of G(0)-arrested cell lines by SIVsmmPBj-derived vectors is independent of the viral accessory proteins Vif, Vpx, Vpr, or Nef. In contrast, for the transduction of primary human monocytes, the Vpx protein proved to be essential. However, trans-complementation of HIV-1 vectors with SIVsmmPBj Vpx did not provide the property of gene transfer into monocytes. Taken together, these data indicate that Vpx is essential for the infection of primary monocytes by SIVsmmPBj. Additionally, further genome functions besides the accessory proteins are required for the particular capacity of SIVsmmPBj in transduction or infection events.
