Abrogation of Ref1 retrovirus restriction in human cells

Abstract

We have previously described postentry restriction of murine leukemia virus in mammals. Here we characterize the block in human cells. Restricted infection kinetics are multiple hit at high virus dose, and restriction is abrogated by preexposure to restricted virus. We hypothesize that restricted capsid can titrate out the restriction factor.

FIG. 1.

Titration curve of N- and B-tropic vectors on human cells. Human TE671 cells were infected with serial dilutions of N-eGFP (▪) and B-eGFP (▴) vectors. The percentages of cells infected were determined 48 h postinfection by FACS. Data are representative of three independent experiments.

FIG. 2.

Abrogation of restriction by preexposure to N-lacZ vector. Panels are side scatter versus fluorescence FACS dot plots of human TE671 cells 48 h after infection with MLV eGFP-encoding vectors. Fluorescence units are arbitrary. The gate used to determine percentages of infected cells is shown (panel i). N- and B-tropic virus titers were equalized on MDTF cells, and the N- and B-lacZ dose was 0.3 MDTF IU/TE671 cell. The N- and B-eGFP dose was ∼8,000 MDTF IU. Cells were infected with N-eGFP vector (panels i, iii, and v) and B-eGFP vector (panels ii, iv, and vi). Cells in panels iii and iv were preexposed to N-lacZ vector for 6 h prior to eGFP vector infection, and panels v and vi were preexposed to B-lacZ vector for 6 h prior to eGFP vector infection. Cells were washed once in medium after exposure to lacZ vectors prior to exposure to eGFP vectors. These data are representative of three independent experiments.

FIG. 3.

Time course of abrogation. TE671 cells were preexposed to abrogating N-lacZ vector (0.1 MDTF IU/TE671 cell) for periods of 1 to 12 h and then infected with a fixed titer of N-eGFP vector (∼8,000 MDTF IU). Infection was measured by FACS 24 h post-N-eGFP infection. Infection of N-eGFP was maximal after 6 h of preexposure to N-lacZ. These data are representative of three independent experiments.

FIG. 4.

Titration of LNCX virus for abrogation of N-eGFP vector restriction. TE671 cells were preexposed to a serial dilution of sixfold-concentrated abrogating N- or B-LNCX virus for 6 h and then infected with a fixed titer of either N- or B-eGFP vector. Cells were analyzed for eGFP expression 48 h later. N- and B-eGFP titers were equalized on MDTF cells, and around 10,000 MDTF IU was used per point. A titer of 10⁵ IU/ml for N-LNCX and B-LNCX was measured on MDTF cells by measurement of CFU after treatment with G418 (1 mg/ml). Data are representative of several experiments performed with LNCX and lacZ abrogating vectors.

FIG. 5.

Titration curve of N-eGFP vector after abrogation of restriction. TE671 cells were infected with serial dilutions of N-eGFP (⧫) and B-eGFP (▪) vectors without preexposure and N-eGFP (▴) and B-eGFP (•) vectors after 6 h of preexposure to an abrogating dose (0.3 MDTF IU/TE671 cell) of N-lacZ vector. Infection was measured 48 h postinfection by FACS. Data are representative of two independent experiments.