Characterization of murine leukemia virus restriction in mammals

Abstract

Restriction of murine leukemia virus (MLV) was examined in cells from a range of mammals. All nonmurine restrictions were saturable blocks to N-tropic MLV infection, and several were prior to reverse transcription. We demonstrate restriction in cells from bat and show that if we express the murine restriction factor Fv1 in human cells, then Fv1, not the human host, defines the stage at which infection is blocked.

FIG. 1.

Titration of N- and B-GFP vectors. A total of 10⁵ cells in 6-well plates were infected with twofold serial dilutions of either N-GFP (•) or B-GFP (▪) vector prepared as previously described. Virus dose was measured in terms of iu on unrestricting MDTF cells. GFP-positive cells were enumerated by FACS 48 h postinfection as previously described (22). Guides with a slope of 2 indicating two-or-greater-hit kinetics are shown, and guides with a slope of 1 indicating single-hit kinetics at low dose are shown in panels for hamster and bat cells. Data are representative of results from three independent experiments.

FIG. 2.

Measurement of DNA synthesis after restricted and unrestricted infection by TaqMan quantitative PCR. Abbreviations: NIH, murine NIH 3T3; H, hamster BHK; Pig, porcine CPK; Cow, bovine MDBK; Bat, Bat Tb-1-lu; AGM, AGM CV1; Hu, human TE671. A total of 10⁵ cells were infected with equal doses of DNase-treated N- or B-GFP vector at a multiplicity of unrestricted infection of 0.15 to 0.25. Viral doses required to achieve these multiplicities were determined independently for each line and are recorded in Table 1. Cells were infected for 6 h, and then total DNA was extracted by DNAeasy (Qiagen, Chatsworth, Calif.). Template copy number values were assigned with reference to a standard curve generated by PCR of plasmid dilutions as previously described (22). One hundred nanograms of DNA was subjected to PCR using primers and probe specific for GFP as previously described (2). Total DNA includes newly synthesized linear and circular molecules as well as integrated provirus. Values of B/N copy numbers are recorded above each pair of columns. Data are representative of results from two independent experiments, and error bars indicate standard errors of the mean.

FIG. 3.

Restriction in human TE671 cells overexpressing Fv1 N (TEN cells). Duplicate samples of 10⁵ cells were infected with 6 × 10³ MDTF iu of DNase-treated N-, B-, and NB-GFP vectors. (A) GFP-positive cells were enumerated by FACS 48 h after infection. Plots are side scatter versus green fluorescence in FL1. The region denoting positive cells and percentage of infection are shown. (B) Quantitative TaqMan PCR was performed, using DNAeasy (Qiagen), on 100 ng of total DNA purified from TEN cells (TE671 overexpressing Fv1 N) 6 h after infection. PCR was performed using primers and probe to GFP as previously described (2). Data are representative of results for two independent TEN clones and two independent experiments. Error bars indicate standard errors of the mean.

FIG. 4.

Phylogenetic distribution of restriction among vertebrates. Underlined species restrict, and species not underlined do not. Cell lines were tested by infecting 10⁵ cells with equal doses of N- and B-GFP vectors as previously described (19). The cell lines were from duck (duck embryo), turkey (Tur8), quail (QT6 and QT35), dunnart (SC300), ferret (MPF), mink (Mv-1-lu), dog, (CF2S⁺L⁻, D17, A72, DK), cat (FEA, AH927, CRFK), horse (NBL6, EDERM), pig (PAE, ST IOWA, SKL, CPK, MPK, PK15), bison (bu IMR-31), cow (MDBK), dolphin (Db-1-tes), mouse (NIH 3T3, BALB/3T12, MDTF), rat (HSN, NRK) golden hamster (BHK), Chinese hamster (CHO, A23), rabbit (SIRC), cotton tail (EREp), bat (Tb-1-lu), owl monkey (OMK), AGM (CV1, Vero), rhesus monkey (LLC-MK2, FRhK4), gibbon (MLA144), squirrel monkey (Pindak), and human (TE671, HeLa, MCF, MRC5, HT1080, NP2, Hos) and were obtained from the Centro Substrati Cellulari, Brescia, Italy, and the American Type Culture Collection. The tree was drawn according to published literature (6, 15, 18). Abbreviations: Mam, mammals; Av, birds; M, marsupials; Eu, Eutheria. Eutherian orders: Ca, Carnivora; Pe, Perissodactyla; A, Artiodactyla; Ce, Cetacea; R, Rodentia; L, Lagomorpha; Ch, Chiroptera; Pr, Primates.