Fig. 8. AGM and human cells do not restrict SIVagmTan infection. AGM (CV-1) and human (TE671) cell lines were infected with serially diluted SIVagmTan reporter virus. The inoculum level is given in TE671 infectious units (i.u.). The percentage of infected (GFP-positive) cells is plotted for each level of SIVagmTan-GFP inoculum.

Fig. 1. Restriction of N-MLV in AGM cell lines but not those of other primates that restrict HIV-1 or SIVmac. Each primate cell line was infected with serially diluted N-GFP or B-GFP vectors. Each vector stock was also titrated on Fv1-null MDTF cells, and the inoculum level is given in MDTF infectious units (i.u.). The percentage of infected (GFP-positive) cells is plotted for each level of N-GFP or B-GFP inoculum.

Fig. 5. Primate lentiviruses do not significantly abrogate Ref1-mediated N-MLV restriction in human cells. Human TE671 cells were inoculated with fixed and equivalent doses [in MDTF infectious units (i.u.)] of N-GFP (filled symbols) or B-GFP (open symbols) in the presence of increasing doses of abrogating HIV-1-Puro (A), HIV-2-Puro (B) or SIV-lacZ (C) particles. Higher doses of N-GFP and B-GFP were used in (B) and (C) than in (A). The percentage of MLV-GFP-infected cells is plotted as a function of abrogating virus dose given as TE671 i.u.

Fig. 6. Abrogation of lentivirus restriction by N-MLV particles. (A) Abrogation of Lv1 restriction in CV-1 but not OMK cells by N-MLV particles. CV-1 and OMK cells were infected with a fixed dose of HIV-1-GFP or SIV-GFP, as indicated, in the presence of increasing doses of abrogating N-Neo (filled symbols) or B-Neo (open symbols) particles. The percentage of HIV-1-GFP- or SIV-GFP-infected cells as a function of abrogating MLV dose is plotted. Abrogating virus dose is given as infectious units (i.u.) as measured on MDTF cells. (B) Abrogation of Lv1 restriction by HIV-1 particles. CV-1 and OMK cells, as indicated, were infected with a fixed dose of HIV-1-GFP or SIV-GFP as in (A) in the presence of the indicated levels (given as ng of p24) of genome-less HIV-1 VLPs. (C) Abrogation of EIAV restriction in human cells by N-MLV particles. TE671 cells were infected with a fixed dose of EIAV-GFP in the presence of increasing amounts of N-Neo (filled symbols) or B-Neo (open symbols) particles. The percentage of EIAV-GFP-infected cells is plotted.

Fig. 2. Saturable resistance to primate lentiviruses and N-MLV in CV-1 cells. (A) HIV-2 is restricted in CV-1 (AGM) but not in human (TE671) cells. CV-1 cells (filled symbols) or TE671 cells (open symbols) were inoculated with a fixed dose of HIV-2-GFP vector in the presence of increasing amounts of abrogating HIV-2-Puro particles. The percentage of HIV-2-GFP-infected cells as a function of abrogating virus dose is plotted. Abrogating virus dose is given in infectious units (i.u.) measured as puromycin-resistant colony formation on TE671 cells. (B) Abrogation of SIV-GFP restriction by HIV-2-Puro particles. CV-1 cells were inoculated with a fixed dose of SIV-GFP in the presence of abrogating HIV-2 particles, as in (A), and the percentage of SIV-GFP infected cells is plotted. (C) Resistance of CV-1 cell to N-GFP is saturable. CV-1 cells were inoculated with a fixed dose of N-GFP in the presence of increasing levels of abrogating N-Neo or B-Neo vector particles. Abrogating virus dose is given as MDTF i.u. as measured by G418-resistant colony formation.

Fig. 3. Abrogation of N-MLV restriction in CV-1 cells by primate lenti virus particles. (A) Abrogation of N-GFP restriction in CV-1 cells by HIV-1 particles. CV-1 cells were inoculated with a fixed and equivalent dose of N-GFP or B-GFP reporter virus (as measured on MDTF cells) in the presence of increasing amounts of abrogating HIV-1-Puro particles. Abrogating virus dose is given as infectious units (i.u.) on TE671 cells. The percentage of N-GFP- (filled symbols) and B-GFP- (open symbols) infected cells is shown. (B) Complete abrogation of N-MLV restriction by HIV-1 particles. N-GFP (filled symbols) and B-GFP (open symbols) were titrated in CV-1 cells, precisely as in Figure 1, except that inoculation was performed in the presence of saturating levels (100 ng of p24) of genome-less HIV-1 VLPs. (C and D) Abrogation of N-MLV restriction by HIV-2 and SIVmac particles. CV-1 cells were inoculated with fixed and equivalent doses of N-GFP (filled symbols) and B-GFP (open symbols), in the presence of increasing amounts of abrogating HIV-2-Puro (C) or SIV-LacZ (D) vector particles. Abrogating virus dose is given as the titer (i.u.) as measured on TE671 cells.

Fig. 7. Phenotypic polymorphism in Lv1 among AGMs. (A) Variation in restriction of HIV-1 and SIVmac in AGM cells. HIV-1-GFP and SIV-GFP stocks were normalized to give nearly identical titration curves on TE671 cells, as shown, and titrated on CV-1, BS-C-1 and Vero cell lines. The percentage of HIV-1-GFP- and SIV-GFP-infected AGM cells for each inoculum dose, in TE671 infectious units (i.u.), is plotted. (B) The CA-p2 region of Gag determines differential restriction of HIV-1 and SIVmac on AGM cell lines. Each AGM cell line was inoculated with equivalent doses of HIV-1-GFP, SIV-GFP and SIV(HIV CA)-GFP, as indicated, normalized using TE671 target cells. The results are given as the percentage of infected AGM cells obtained, divided by the percentage of infected TE671 cells. (C) Abrogation of N-MLV restriction by HIV-1 and SIVmac VLPs on AGM cells. Each AGM cell line was inoculated with a fixed dose of N-MLV-GFP in the presence of increasing levels (given in ng of p24 or p27) of abrogating HIV-1 or SIVmac VLPs. The fold increase in the number of infected N-GFP cells as a function of abrogating VLP dose is plotted.

Fig. 4. Restriction of EIAV in human and AGM cells by the same factors that restrict primate lentiviruses and N-MLV. (A) Non-linear titration curve upon infection of human (TE671) cells by EIAV. TE671 cells were inoculated with an increasing dose of EIAV-GFP. The diagonal guide line indicates a slope of 1. (B) Abrogation of EIAV restriction in TE671 cells by EIAV particles. TE671 cells were inoculated with a fixed dose of EIAV-GFP in the presence of increasing levels of EIAV VLPs. (C) Abrogation of N-MLV restriction in TE671 cells by EIAV particles. TE671 cells were inoculated with fixed and equivalent doses of N-GFP or B-GFP in the presence of increasing levels of EIAV VLPs (given in ml of transfected cell supernatant). (D) Non-linear titration curve upon infection of CV-1 cells by EIAV. CV-1 cells were inoculated with an increasing dose of EIAV-GFP, and the percentage of infected cells is plotted. (E and F) Abrogation of EIAV restriction in CV-1 cells by EIAV and HIV-1 particles. CV-1 cells were inoculated with a fixed dose of EIAV-GFP in the presence of increasing levels of (E) EIAV VLPs or (F) HIV-1-Puro particles (given in ng of p24). The percentage of EIAV-GFP infected cells as a function of abrogating particle dose is plotted. (G) Abrogation of N-MLV restriction in CV-1 cells by EIAV particles. CV-1 cells were inoculated with fixed and equivalent doses of N-GFP (filled symbols) or B-GFP (open symbols), as determined by titration on MDTF cells, in the presence of increasing levels of EIAV VLPs. The percentage of MLV-GFP-infected cells is plotted. In the absence of a convenient assay for measuring EIAV VLP concentration, a single stock of EIAV VLP-containing supernatant was used in each of the experiments presented herein, and the VLP level is given as a volume (ml) of this stock. However, each experiment was performed at least twice with independent VLP stocks, with similar results.