Article Figures & Data
Figures
- Fig. 1.
Processing ability of various rodent cell lines.M. musculus (MGT5.CyT, EL4.CyT, and L1-2.CyT), M. dunni (MDTF.CyT), hamster (CHO.CyT), or rat (Rat2.CD4.R5) cell lines with or without transfected hu-cyclin T1 were infected with NL4-3(VSV-G) at an MOI of 1. (A) Immunoblot analysis of infected cell lysates and virions pelleted from supernatant with anti-HIV serum. (B) Virion production of the infected cell lines. (C) Infectious titer of virus produced by the infected cell lines. Results are the average of triplicates. (D) Calculated ratio of TCID50 to supernatant p24gag from panels B and C.
- Fig. 2.
Electron microscopy of HIV-1 infected CHO and MDTF derivatives. (Upper panels) (a) Ultrathin sections were processed by glutaraldehyde-osmium fixation and ERL embedding. Budding structures (a) and extracellular HIV-1 particles are present. (b and c) Immunoelectron microscopy with anti-CA antibody of HIV-1-infected CHO derivative shows labeled budding structures (b) and adjacent extracellular particles (c). Gold particles are scarce in the cytoplasm and nearly absent in the extracellular space. (d and e) Gold-labeled HIV-like particles in the ER cisternae. Some cytoplasmic vacuoles (V) were labeled (arrow), while adjacent vacuoles remained unlabeled (arrowhead). (Lower panels) (a) Ultrathin sections of infected, fixed, and embedded MDTF.CD4.R5.CyT cells at low magnification showed virus particles at the plasma membrane. Morphological alterations were not observed. (b) Higher magnification revealed mature extracellular HIV-1 particles adjacent to the plasma membrane. (c) The extracellular particles were immunolabeled.
- Fig. 3.
CHO.CD4.R5.CyT but not other rodent cells support low-level HIV-1 replication. (A) MGT5.CyT, CHO.CD4.R5.CyT, MDTF.CD4.R5.CyT, LT4.R5.CyT, and HOS.CD4.R5 cells were infected with HXB.BaL and CHO.CD4.R5.CyT and HOS.CD4.R5 cells were also infected with NL-BaL at an MOI of 0.05, and p24gag production was measured over time. (B) CHO.CD4.R5.CyT and HOS.CD4.R5 were infected with HIV.YU2.GFP at an MOI of 0.05, and the percentage of green cells was scored by fluorescence-activated cell sorting analysis.
- Fig. 4.
CHO cells are competent for coreceptor function but fail to support efficient virus replication. CHO.CD4.R5.CyT and HOS.CD4.R5 cells were infected with JR.FL pseudotyped firefly luciferase or VSV-G pseudotyped Renilla luciferase single-cycle reporter viruses. Luciferase activity was measured in cell lysates 3 days postinfection. The results are the averages of triplicates and are representative of three experiments.
- Fig. 5.
The assembly defect in 3T3 cells is complemented by fusion to human cells. (A) Strategy for testing HIV assembly and production in mouse-human heterokaryons. MGT5.CyT, GHOST.R5, and MDTF.CD4.R5.CyT cells were infected at an MOI of 1 with NL4-3(VSV-G), washed three times, and fused 3 days later to 293T, 3T3, or MDTF cells. After 24 h postfusion, the cells were lysed for immunoblot analysis, and virions were collected for p24gag and TCID50 measurement. To control for input virus contamination, mock fusions in which PEG was omitted were analyzed in parallel. (B) p24gagconcentration in heterokaryon supernatants. (C) TCID50measurement of virus in heterokaryon supernatants. (D) TCID50 measurement of virus in M. dunni-human heterokaryon supernatants. (E) Immunoblot analysis of heterokaryon lysates using anti-HIV serum.
- Fig. 6.
Fusing human cells to infected murine cells that express hu-cyclin T1 increases LTR transactivation. Murine or human cells were infected with luciferase reporter virus. Three days postinfection, the cells were cocultivated with JR.FL Env-expressing or control cells for an additional 24 or 48 h to allow Env-mediated fusion between the two cell types. Luciferase activity in the cell lysates was then measured. Results are the averages of triplicates and are representative of three experiments.
