Embryonic stem cells use ZFP809 to silence retroviral DNAs

Abstract

Embryonic stem cells (ESCs) and other primitive stem cells of mice have been known for more than 30 years to potently block retrovirus replication. Infection of ESCs by the murine leukaemia viruses (MLVs) results in the normal establishment of integrated proviral DNA, but this DNA is then transcriptionally silenced, preventing further viral spread. The repression is largely mediated by trans-acting factors that recognize a conserved sequence element termed the primer binding site, an 18-base pair sequence complementary to the 3' end of a cellular transfer RNA. A specific tRNA is annealed to the primer binding site sequence of the viral genomic RNA, and is used to prime DNA synthesis. This same sequence in the context of the integrated proviral DNA is targeted for silencing in ESCs. We have recently shown that a large protein complex binding to the primer binding site in ESCs contains TRIM28 (refs 8, 9), a well-characterized transcriptional co-repressor. An important question remains as to the identity of the factor that directly recognizes integrated retroviral DNAs and recruits TRIM28 to mediate their specific silencing. Here we identify the zinc finger protein ZFP809 as the recognition molecule that bridges the integrated proviral DNA and TRIM28. We show that expression of ZFP809 is sufficient to render even differentiated cells highly resistant to MLV infection. Furthermore, we demonstrate that ZFP809 is able to potently block transcription from DNA constructs of human T-cell lymphotropic virus-1 (HTLV-1), which use the same primer tRNA. These results identify ZFP809 as a DNA-binding factor that specifically recognizes a large subset of mammalian retroviruses and retroelements, targeting them for transcriptional silencing. We propose that ZFP809 evolved as a stem-cell-specific retroviral restriction factor, and therefore constitutes a new component of the intrinsic immune system of stem cells.

Figure 1. Identification of candidate PBS^Pro-DNA binding factors

(A) F9 EC cell nuclear extract prepared and incubated with radiolabeled probes corresponding to PBS^Pro (PRO), PBS^B2 (B2) or PBS^Pro with a 15 bp extension (PRO+15bp). Crosslinking was performed by exposure to UV light. Competition was achieved through the addition of 100 pmols of unlabeled probe corresponding to either PBS^Pro or PBS^B2. (B) Crosslinked nuclear extracts were subjected to immunoprecipitation with anti-FLAG or two separate anti-TRIM28 antibodies. (C) Purification scheme used to prepare samples for identification by mass spectrometry. (D) SDS PAGE of samples prepared by purification scheme outlined above, visualized by Coomassie stain. Bands excised for protein identification by mass spectrometry are labeled “A” and “B”.

Figure 2. Reconstitution of the PBS^Pro binding complex with recombinant proteins

(A) 50 ng or 250 ng of recombinant GST-tagged ZFP124, L1td1 and ZFP809 added to EMSA reactions with radiolabeled labeled probes corresponding to PBS^Pro (PRO) or PBS^B2 (B2). (B) EMSAs performed as above, with PCC4 EC nuclear extract or 25, 50, or 100 ng of GST-ZFP809 protein and 100 ng, 200 ng or 500 ng of GST-TRIM28 of protein as indicated.

Figure 3. Stable expression of ZFP809 in 293A cells leads to reconstitution of the PBS silencing complex in vivo

(A) Nuclear extracts from clonal cell lines control Clone 1 (empty vector control), Clone 5 (cell line stably expressing FLAG-tagged ZFP809(1-353) or Clone 9 (cell line generated with same construct as Clone 5 but which does not express ZFP809) probed by western blot with antisera for FLAG (upper panel), TRIM28 (middle panel), or beta-actin (lower panel). (B) Nuclear extracts prepared either from PCC4 EC cells or cell lines control Clone 1, Clone 9, or Clone 5 used in EMSA reactions with radiolabeled probes corresponding to PBS^Pro (PRO) or PBS^B2 (B2). In final three lanes the PBS silencing complex is supershifted by the addition of 2 µg of either anti-TRIM28, anti-FLAG, or a control anti-HA antibody. (C) ZFP809(1-353) and TRIM28 co-immunoprecipitate in DNase treated Clone 5 whole cell extracts in the presence of ethidium bromide. Control immunoprecipitations were performed with of HA antisera. Immunoprecipitated ZFP809(1-353) and Co-immunoprecipitated TRIM28 was detected by western blot.

Figure 4. Expression of ZFP809 in a differentiated cell line causes a potent block to the replication of PBS^Pro utilizing retroviruses

(A) 293A, control Clone 1, Clone 9 and Clone 5 cell lines were infected with either PBS^Pro or PBS^B2 VSV-G pseudotyped MLV virions expressing the puromycin resistance gene. Infection efficiency in each cell line was monitored by colony count after of puromycin selection. Graph shows ratio of B2/PRO infection efficiency in each cell line, normalized with 293A =1. Error bars show +/− standard error, with n=3. (B) Control Clone 1, Clone 9 and Clone 5 cell lines were infected at a low multiplicity with replication-competent amphotropic MLV utilizing either a PBS^Pro (PRO) or PBS^Q (Q). Viral spread in these cell lines was monitored by analysis or reverse transcriptase activity in the culture media every day for 6 days. (C) HTLV-1 LTR or HIV-1 LTR firefly luciferase reporter activity monitored in cell lines control Clone 1, Clone 5, and Clone 9 which were simultaneously transfected with increasing amounts of a Tax expressing vector (pTAX) or TAT expressing vector (pTAT-HA) as shown. Firefly luciferase values normalized to renilla luciferase values from a simultaneously transfected renilla control vector. Error bars show +/− standard error, with n=3.