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Figure 4 | Retrovirology

Figure 4

From: IFITM proteins are incorporated onto HIV-1 virion particles and negatively imprint their infectivity

Figure 4

IFITMs are interferon-regulated proteins that display heterogeneous cell type dependent expression and appear incorporated in HIV-1 virions proportionally to their intracellular levels. A) The ectopic expression levels of IFITMs in HEK293T cells used before were compared to the endogenous expression of IFITMs in different cells, incubated or not with IFNα (at 1000 U/mL for 24 hours). Given that the antibodies in our hands did not distinguish between IFITM members anti-IFITM1, -2 and -3 antibodies were used together, so that all three forms are recognized. B) HEK293T and HeLaP4 cells were transfected with DNAs coding HIV-1-gfp and twenty-four hours later cells were stimulated with 1000U/mL of IFNα for further two days. Virions were then purified and analyzed by WB and upon exo-RT normalization they were used to challenge HeLa P4 cells. C) Primary macrophages obtained upon differentiation of monocytes in M-CSF for 4 days were challenged with an MOI of 0,1 of the R5-tropic HIV-1 strain ADA. Aliquots of the supernatant were harvested every few days and the extent of viral spread through the culture was measured by exo-RT activity. Cells and virion particles were harvested at the indicated time points after infection. Virion particles obtained from day 6 and 9 were normalized by exo-RT activity, while the amount of virus obtained at day 3 was too low to be quantified further. Exo-RT normalized virions obtained at day 6 and 9 were used to challenge HeLaP5 cells for a MAGI assay. The graph presents a typical replication curve obtained upon infection of ADA in primary MDM and the dotted line represents the limit of detection of the assay. All WB panels present representative results obtained out of 3 to 5 independent experiments and donors, while the graphs present averages and SEM of 3 independent experiments.

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