(A) The viral proteins HIV-1 Vpr, SIVsmmPBj1.9 Vpx and related fusion proteins encoded by the expression vector pcDNA3.1. For generation of the fusion protein, the full length Vpx was ligated to HIV-1 Vpr. HA, hemagglutinin tag. (B) HIV-1- or SIVsmmPBj- derived transfer vectors coding for EGFP. In the PBj4xko-EGFP vector, coding regions of all four accessory genes are knocked-out by insertion of stop-codons marked by an asterisk. (C) PBj- and HIV-1-derived packaging constructs coding for Gag-Pol and the regulatory proteins Tat and Rev. These constructs were also used for production of virus-like particles. For pseudotyping of vectors or virus-like particles, the vesicular stomatitis virus G protein (VSV-G, not shown) was used. (D) Western blots of supernatants or lysates of 293T packaging cells cotransfected with the respective vector constructs required for generation of VSV-G-pseudotyped lentiviral vectors (HIV-1 LV or PBj4xko LV), and one of the expression constructs encoding Vpr, Vpx or the Vpr/Vpx fusion protein as indicated on the top. As controls, supernatants and lysates of cells transfected only with one of the Vpr, Vpx or Vpr/Vpx constructs were analyzed. Cell supernatants were purified by filtering and sucrose cushion centrifugation. For labelling, antibodies directed against HA, Vpx, or the envelope protein VSV-G were used. Ψ, packaging signal; LTR, long terminal repeat; SD, splice donor; cPPT, central polypurine tract; RRE, rev responsive element; SA, splice acceptor; CMV, cytomegalovirus promoter; BGHpA, bovine growth hormone polyadenylation signal.

(A) Packaging of Vpx into PBj-derived VLPs was detected by Western-blotting. VLPs were generated by cotransfection of the PBj packaging vector pPBj-psi10 and the VSV-G and Vpx expression plasmids. Forty-eight hours post transfection, vector containing supernatant was collected from the 293T cells, filtered and purified twice by ultracentrifugation through a sucrose cushion. Purified VLPs were analyzed on Western blots using antibodies directed against Vpx or the envelope protein VSV-G. (B, C) Transduction of primary human monocytes or monocyte-derived cells 2 h after pre-incubation with Vpx-containing or non-containing PBj-derived VLPs (VLP(PBj)) at an MOIeq of 1 (determined by RT-Assay with standards of known infectivity) as indicated. Percentage of EGFP-expressing cells transduced at various days after isolation using (B) HIV-1 vectors or (C) PBj4xko-EGFP vectors. The percentage of transduced cells was determined by flow cytometry 6 days post transduction. The means of two different donors are shown. (D) Influence of Vpx on activation and differentiation of monocytes or monocyte-derived cells. At the indicated day after isolation, cells were incubated for 48 h with VLPs supplemented with Vpx (+VLPs +Vpx) or not (+VLPs −Vpx) at an MOIeq of 1, in comparison with untreated cells (−VLPs). The surface expression of the markers indicated was determined by FACS analysis. One representative donor out of three is shown.

(A) Transduction of simian monocytes. Monocytes from Rhesus or Pig-tailed macaques were pre-incubated for 2 h with PBj-derived VLPs either containing or lacking Vpx at an MOIeq of 1, and subsequently transduced with HIV-1 or PBj4xko-EGFP vectors at an MOI of 7 as indicated. Means of three animals of each species are shown. (B) Replication kinetics of HIV-1 or SIVsmmPBj. Monocytes of Pig-tailed macaques were stimulated at day 1 post isolation for 8 h with TPA before incubation with VLPs as indicated at an MOIeq of 1, and subsequent infected an MOI of 0.5 with HIV-1 strain SF165 or SIVsmmPBj1.9. Virus replication was assessed by quantifying RT antigen in cell culture supernatants. Means of three different donors out of two independent experiments are shown.

Percentage of EGFP-expressing monocytes or monocyte-derived cells transduced at various days post isolation using VSV-G pseudotyped vector particles (MOI 10) supplemented with Vpx, Vpr, or the Vpr/Vpx fusion protein as indicated. The percentage of EGFP-expressing cells was determined by flow cytometry 6 days post transduction. (A) Transduction efficiency of PBj4xko-EGFP-derived vector particles. (B) Transduction efficiency of HIV-1-derived vector particles. Positive control: PBΔE-EGFP-derived vector containing all accessory genes. Means of three different donors are shown.

Percentage of EGFP-expressing cells 6 days post transduction. (A) Monocytes (day 2 post isolation) were pre-incubated for 2 h with increasing amounts of PBj-derived VLPs (VLP(PBj)) as indicated, and transduced with HIV-1 vectors at an MOI of 10. (B) Monocytes were incubated with PBj-derived VLPs at an MOIeq of 1 (determined by RT-Assay with standards of known infectivity), followed by transduction with HIV-1 vectors at increasing MOI as indicated. The means of two different donors are shown. (C) On day 1 post isolation, monocytes were incubated for 2 h with Vpx-containing or non-containing VLPs at an MOIeq of 1 and subsequently infected with CCR5-tropic HIV-1 strain SF165 at an MOI of 0.5. SIVsmmPBj1.9 infection was performed without previous VLP incubation. Replication kinetics of HIV-1 or SIVsmmPBj after pre-incubation of monocytes with VLPs as indicated. Virus replication was assessed by quantifying RT activity in cell culture supernatants. Means of two different donors from one representative out of three experiments are shown.