Murine leukemia viruses (MLVs) and related retroelements are potently restricted in embryonic cells by postintegration transcriptional silencing, likely to protect the germ line from insertional mutagenesis. This silencing is in large part attributable to the presence of a nuclear repression complex, which targets a sequence element of the proviral DNA, the repressor-binding site. The repressor-binding site closely overlaps the tRNA primer binding site, a highly conserved sequence essential for virus replication and defining the site of initiation of DNA synthesis during reverse transcription. We have recently demonstrated that the cellular corepressor TRIM28 is recruited to the proline tRNA primer-binding site used by many MLVs and is required to mediate this silencing. Here, we show that TRIM28 is also required for the restriction of retroviruses using a completely distinct tRNA for the priming of their DNA synthesis, namely Lys-1,2 tRNA. These results generalize the role of TRIM28 in retroviral restriction and suggest that this system has evolved to restrict multiple retroviruses.urine leukemia viruses (MLVs) are unable to replicate in embryonic cells (1-3). The block to replication occurs after the viral DNA has integrated into the host genome and is caused by transcriptional silencing of the proviruses (2, 4). This silencing is the result of several cumulative effects that include poor enhancer function of the 5Ј LTR (5-7), the binding of several cell-type specific transacting transcriptional repressors (6, 8), and de novo DNA methylation (4). A critical target of this repression is the DNA element known as the repressor binding site (RBS) (2, 5, 9, 10). The RBS element comprises 17 bp that overlap closely with the 18-bp primer binding site (PBS) of MLV (2), a sequence complementary to the cellular proline tRNA (tRNA Pro ) used to prime minus-strand DNA synthesis during reverse transcription (11). This silencing can be abrogated by a single base-pair substitution in the PBS known as the B2 mutation (2). Characterization of RBS-mediated restriction demonstrated that the 17-bp sequence was able to induce transcriptional silencing of reporter constructs in EC cells independently of position and orientation of the RBS sequence (10, 12). Furthermore, exonuclease III protection assay and EMSA experiments revealed the presence of a DNA-binding activity that is specifically enriched in stem cell nuclear extracts (10, 12). Based on these experiments, a trans-acting DNA-binding factor was postulated to exist; we have recently demonstrated that a high molecular weight complex binds the RBS sequence, and that a key component of this complex is the TRIM28 protein (13). TRIM28 (also known as Kap-1, or Tif1-beta) is a well characterized transcriptional corepressor that is recruited to its target genes by interactions with the Krüppel associated box (KRAB) zinc-finger DNA-binding proteins (14). This interaction is mediated via the KRAB box domain and leads to these DNA-binding proteins, serving as sequence-specific transcr...