Primer binding site-dependent restriction of murine leukemia virus requires HP1 binding by TRIM28

Abstract

TRIM28 is a transcriptional corepressor which is required for primer binding site (PBS)-dependent restriction of murine leukemia virus (MLV) replication in embryonic stem and embryonic carcinoma (EC) cells. PBS-dependent restriction of MLV leads to transcriptional silencing of the integrated provirus and has been shown to correlate with TRIM28-mediated recruitment of HP1 to the silenced loci. Here we show, using a cell line with a point mutation in the HP1 binding domain of TRIM28, that interaction with HP1 is absolutely required for the PBS-dependent restriction of MLV in the F9 EC cell line.

FIG. 1.

(A) Nuclear extracts from F9, PCC4, PCC4 SCRAM, PCC4 RNAi TRIM28 (111) (20), HeLa, NIH 3T3, RAT2, and 293A cells were prepared and used in EMSA reaction mixtures with a ³³P-labeled 28-bp probe corresponding to either the WT (PRO) or the mutated B2 MLV PBS sequence, as indicated. RBS, repressor binding site. (B) Western blots of the same nuclear extracts probed with anti-TRIM28 ab22553 (ABCAM) and anti-β-actin antibodies.

FIG. 2.

(A) Nuclear extracts from F9, TRIM28^+/−, and TRIM28^HP1box/− cell lines were prepared and used in EMSAs with a ³³P-labeled 28-bp probe corresponding to the WT MLV PBS. In the last three lanes, EMSAs were performed with the addition of 3 μg of anti-TRIM28 antibody ab22553 (ABCAM). RBS, repressor binding site. (B) Western blots of the same nuclear extracts probed with anti-TRIM28 and anti-β-actin antibodies.

FIG. 3.

(A) Repression of B2 versus that of PRO MLV. RAT2, F9, TRIM28^+/−, and TRIM28^HP1box/− cell lines were infected with VSV-G-pseudotyped MLV containing either LJ-PAdMLPEnh− (WT) or LJB2-ADMLPEnh− (B2) constructs. Infection efficiency in each cell line was monitored by colony counting after 2 weeks of G418 selection. The graph shows ratios of B2 to WT infection efficiency in each cell line, normalized with RAT2, defined as equal to 1. Error bars show ± standard errors (n = 5). (B) F9, TRIM28^+/−, and TRIM28^HP1box/− cell lines were infected with either WT or B2 VSV-G-pseudotyped MLV as described above. Two weeks later, genomic DNA was extracted from these cells and PCR was performed using primers specific for either the MLV PBS or GAPDH (glyceraldehyde-3-phosphate dehydrogenase), as described previously (20).