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NP220 mediates silencing of unintegrated retroviral DNA

Abstract

The entry of foreign DNA into many mammalian cell types triggers the innate immune system, a complex set of responses to prevent infection by pathogens. One aspect of the response is the potent epigenetic silencing of incoming viral DNAs1, including the extrachromosomal DNAs that are formed immediately after infection by retroviruses. These unintegrated viral DNAs are very poorly transcribed in all cells, even in permissive cells, in contrast to the robust expression that is observed after viral integration2,3,4,5. The factors that are responsible for this low expression have not yet been identified. Here we performed a genome-wide CRISPR–Cas9 screen for genes that are required for silencing an integrase-deficient MLV–GFP reporter virus to explore the mechanisms responsible for repression of unintegrated viral DNAs in human cells. Our screen identified the DNA-binding protein NP220, the three proteins (MPP8, TASOR and PPHLN1) that comprise the HUSH complex—which silences proviruses in heterochromatin6 and retrotransposons7,8—the histone methyltransferase SETDB1, and other host factors that are required for silencing. Further tests by chromatin immunoprecipitation showed that NP220 is the key protein that recruits the HUSH complex, SETDB1 and the histone deacetylases HDAC1 and HDAC4 to silence the unintegrated retroviral DNA. Knockout of NP220 accelerates the replication of retroviruses. These experiments identify the molecular machinery that silences extrachromosomal retroviral DNA.

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Fig. 1: CRISPR–Cas9 screen to identify host factors responsible for silencing of unintegrated retroviral DNA.
Fig. 2: NP220, HUSH complex and SETDB1 are required for the silencing of unintegrated MLV DNA.
Fig. 3: NP220 recruits the HUSH complex and SETDB1 to silence unintegrated MLV DNA.
Fig. 4: NP220 recruits HDAC1 and HDAC4 to deacetylate histone H3 on unintegrated retroviral DNA.
Fig. 5: The binding specificity of NP220 to unintegrated retroviral DNA.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source Data is available for the Figs 3a–g, 4c–h, 5b, d and Extended Data Figs. 1e–j, 3a–e, 4a–d, 6f, g. For gel source data, see Supplementary Fig. 1.

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Acknowledgements

Cell sorting was performed with the assistance of A. Figueroa in the Core Facility of Department of Microbiology and Immunology at Columbia University. This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health (NIH) grant R01 CA30488 (to S.P.G.).

Reviewer information

Nature thanks A. Engelman, M. Lorincz, L. Naldini and M. Ott for their contribution to the peer review of this work.

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Affiliations

Authors

Contributions

Y.Z., G.Z.W. and S.P.G. designed the experiments. Y.Z. and S.P.G. wrote the manuscript, with input from G.Z.W. and O.C. Y.Z. and G.Z.W. performed experiments in Fig. 2 and ChIP–qPCR experiments. All other experiments were performed by Y.Z.

Corresponding author

Correspondence to Stephen P. Goff.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Silencing of unintegrated, but not integrated, retroviral DNA.

a, b, Silencing of unintegrated retroviral DNA. HeLa cells were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) or integrase-deficient (IN(D184A)) MLV-Luc viruses. Total viral DNA levels (a) and luciferase activities (b) were measured 40 h after infection. The viral DNA concentration and luciferase activity of IN(WT) MLV-Luc were set to 1. Data are mean ± s.d.; n = 3 independent experiments. c, d, Silencing of unintegrated retroviral DNA is dependent on histone deacetylation. HeLa (c) and NIH3T3 (d) cells were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) or integrase-deficient (IN(D184A)) MLV-Luc viruses and treated with DMSO (TSA−) or 1 μM HDAC inhibitor trichostatin A (TSA+). Luciferase activities were measured 40 h after infection. Data are mean ± s.d.; n = 3 independent experiments. ej, Repressive epigenetic marks are present on unintegrated retroviral DNA, whereas active epigenetic marks are present on integrated retroviral DNA. HeLa cells were infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) (eg) or integrase-proficient (IN(WT)) (hj) MLV-Luc viruses. At 40 h after infection, ChIP—using antibodies against pan-acetyl H3, H3K9me3 or H3K27me3—followed by qPCR using indicated primers, was performed to assess H3ac (e, h), H3K9me3 (f, i) and H3K27me3 (g, j) modifications across the LTR of unintegrated and integrated MLV-Luc DNA. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. ns, P > 0.05; *P < 0.05; **P < 0.01. P values are from paired two-sided Student’s t-tests. Exact P values are included in the Source Data associated with this figure. k, Knockdown of indicated genes has no effect on viral DNA levels. HeLa cells were first transfected with the indicated siRNA and then infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. Viral DNA levels were measured 40 h after infection. DNA levels in HeLa cells transfected with a non-targeting (NT) control siRNA was set to 1. Data are mean ± s.d.; n = 3 independent experiments. Source data

Extended Data Fig. 2 NP220 and SETDB1 are required for the silencing of unintegrated MLV DNA.

a, b, HeLa (a) or NIH3T3 (b) cells were transfected with indicated siRNAs and then infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. Luciferase activities were measured 40 h after infection and luciferase activity in non-targeting (NT) control siRNA-transfected cells was set to 1 (top). Data are mean ± s.d.; n = 3 independent experiments. The expression of NP220 was determined by western blot (bottom). c, HeLa cells were first transfected with the indicated siRNAs targeting histone methyltransferases and then infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. Luciferase activities were measured 40 h after infection and luciferase activity in non-targeting (NT) control siRNA-transfected HeLa cells was set to 1. Data presented are mean ± s.d.; n = 3 independent experiments. d, Knockdown efficiency of siRNAs used in c. HeLa cells were transfected with the indicated siRNAs targeting histone methyltransferases and mRNA levels of siRNA-targeted genes were measured by qPCR with reverse transcription (RT–qPCR). mRNA levels in non-targeting (NT) control siRNA-transfected HeLa cells was set to 1. Data are mean ± s.d.; n = 3 independent experiments.

Extended Data Fig. 3 NP220 recruits the HUSH complex and SETDB1 to silence unintegrated MLV DNA.

a, b, HA-tagged NP220, NP220 with zinc finger deletion (ΔZnF) (a) or indicated fragments of NP220 (b) were introduced into NP220 knockout HeLa cells and then immunoprecipitated using an HA antibody. Co-immunoprecipitated MPP8 was analysed by western blot. Images are representative of two independent experiments with similar results. c, Parental wild-type (WT) HeLa cells, NP220 knockout HeLa cells and NP220 knockout HeLa cells that were reconstituted with indicated variants of NP220 were subsequently infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. At 40 h after infection, ChIP was performed to assess the association of NP220 across the LTR of unintegrated MLV-Luc DNA. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. ns, P > 0.05; *P < 0.05. P values are from paired two-sided Student’s t-tests. Exact P values are included in the Source Data associated with this figure. d, Parental HeLa cells, MPP8 knockout HeLa cells and MPP8 knockout HeLa cells that were reconstituted with indicated variants of MPP8 were infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. At 40 h after infection, ChIP was performed to assess the association of MPP8 with the LTR of unintegrated MLV-Luc DNA. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. ns, P > 0.05; *P < 0.05. P values are from paired two-sided Student’s t-tests. Exact P values are included in the Source Data associated with this figure. e, HeLa cells were infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. At 40 h after infection, ChIP was performed using the indicated antibodies followed by qPCR using primers targeting 2LTR circles. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. Source data

Extended Data Fig. 4 Interaction between NP220 and HDACs.

a, Screen for interactions between NP220 and HDACs. Endogenous NP220 were immunoprecipitated (IP) from indicated HeLa cell lines and the indicated co-immunoprecipitated HDAC proteins and MPP8 were analysed by western blot using specific antibodies. MPP8 serves as a positive control. Images are representative of two independent experiments with similar results. b, NP220–MPP8 or NP220–HDAC4 interactions are independent of DNA or RNA. Cell lysates from indicated HeLa cell lines were treated with benzonase, DNase or RNase A, endogenous NP220 were immunoprecipitated and co-immunoprecipitating proteins were analysed by western blot using specific antibodies. Images are representative of two independent experiments with similar results. c, d, Interrelationship of histone deacetylation and H3K9 trimethylation on unintegrated viral DNA. c, HeLa cells were infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus and treated with DMSO (TSA−) or 1 μM HDAC inhibitor trichostatin A (TSA+). d, Parental HeLa cells and SETDB1 knockout HeLa cells (SETDB1 KO) were infected with VSV-G pseudotyped, integrase-deficient (IN(D184A)) MLV-Luc virus. At 40 h after infection, ChIP was performed using the indicated antibodies followed by qPCR using primers targeting LTR. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. *P < 0.05; **P < 0.01. P values are from paired two-sided Student’s t-tests. Exact P values are included in the Source Data associated with this figure. Source data

Extended Data Fig. 5 NP220 mediates silencing of unintegrated retroviral DNA from HIV-1 and MPMV, but not RSV.

a, Parental MT-4 cells and NP220 knockout MT-4 cell line (NP220 KO) were infected with VSV-G pseudotyped, integrase-deficient (IN(D64A)) HIV-1 vector NL4.3-Luc. Luciferase activities were measured 40 h after infection and luciferase activity in parental (wild-type) MT-4 cells was set to 1 (top). Data are as mean ± s.d.; n = 3 independent experiments. The expression of NP220 was determined by western blot (bottom). b, COS-7 cells were first transfected with indicated siRNAs and then infected with VSV-G pseudotyped, integrase-deficient (IN(D127A)) MPMV vector SARM-Luc. Luciferase activities were measured 40 h after infection and luciferase activity in non-targeting (NT) control siRNA-transfected cells was set to 1 (top). Data are mean ± s.d.; n = 3 independent experiments. The expression of NP220 was determined by western blot (bottom). ce, Indicated HeLa cell lines were infected with VSV-G pseudotyped, integrase-deficient HIV-1 vector NL4.3-Luc (c), MPMV vector SARM-Luc (d) or RSV vector RCAS-Luc (e). Luciferase activities were measured 40 h after infection. Luciferase activity in parental HeLa cells was set to 1. Data are mean ± s.d.; n = 3 independent experiments. f, g, HDAC1 and HDAC4 are involved in silencing of unintegrated HIV-1 and MPMV DNA. HeLa cells were first transfected with the indicated siRNAs and then infected with VSV-G pseudotyped, integrase-deficient (IN(D64A)) HIV-1 vector NL4.3-Luc (f) or VSV-G pseudotyped, integrase-deficient (IN(D127A)) MPMV vector SARM-Luc (g). Luciferase activities were measured 40 h after infection and luciferase activities in HeLa cells transfected with control non-targeting (NT) siRNA were set to 1. Data are mean ± s.d.; n = 3 independent experiments.

Extended Data Fig. 6 The binding specificity of NP220 bound to unintegrated MLV DNA.

a, The sequence of MLV U3 region. Putative NP220-binding sites are indicated in red. The sequence of the 84-nucleotide probe used for EMSA is italicized and underlined. b, c, Parental or NP220 knockout HeLa cells were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) (b) or integrase-deficient (IN(D184A)) (c) MLV-Luc viruses bearing the indicated deletions or mutations in the U3 region. Luciferase activities were measured 40 h after infection. Data are mean ± s.d.; n = 3 independent experiments. d, e, Indicated HeLa cell lines were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) MLV-Luc virus. d, At the indicated times after infection, luciferase activities were measured. e, Fold increase (KO/WT) was calculated as the ratio of the luciferase activities of knockout cells compared to the luciferase activities of wild-type cells. Data are mean ± s.d.; n = 3 independent experiments. f, g, The dynamics of H3Ac deposition and NP220 association on viral DNA during the course of MLV infection. HeLa cells were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) MLV-Luc virus. At the indicated time points after infection, ChIP was performed using antibodies against H3Ac (f) and NP220 (g) followed by qPCR using primers targeting the LTR, to monitor the association of H3Ac (f) and NP220 (g) with viral DNA. qPCR data from each ChIP were calculated as the percentage of input DNA. Data are mean ± s.d.; n = 3 independent experiments. ns, P > 0.05; *P < 0.05; **P < 0.01. P values are from paired two-sided Student’s t-tests. Exact P values are included in the Source Data associated with this figure. Source data

Extended Data Fig. 7 The binding specificity of NP220 bound to unintegrated HIV-1 DNA.

a, b, The sequences and locations of putative NP220-binding sites in HIV-1 U3 region. Putative NP220-binding sites are indicated in red. c, d, Parental or NP220 knockout HeLa cells were infected with VSV-G pseudotyped, integrase-deficient (IN(D64A)) MLV-Luc viruses bearing indicated deletions in the U3 region. c, Luciferase activities were measured 40 h after infection. d, Fold increase (NP220 KO/NP220 WT) was calculated as the ratio of the luciferase activities of knockout cells compared to the luciferase activities of wild-type cells. Data are mean ± s.d.; n = 3 independent experiments. e, Parental or NP220 knockout HeLa cells were infected with VSV-G pseudotyped, integrase-proficient (IN(WT)) HIV-Luc viruses bearing the indicated deletions in the U3 region. Luciferase activities were measured 40 h after infection. Data are mean ± s.d.; n = 3 independent experiments. f, Knockout of NP220 increases the rate of HIV-1 spreading. g, Parental and NP220 knockout MT-4 cells were infected with HIV-1(NL4.3). Viral spreading was monitored by assay for p24 concentration in the culture medium. Data are mean from two technical ELISA replicates and are representative of two independent experiments.

Extended Data Fig. 8 Schematic of the silencing of unintegrated retroviral DNAs.

Retroviral infection results in the synthesis of a linear double-stranded DNA in the cytoplasm, which is delivered into the nucleus to give rise to two circular forms and the integrated provirus (top). The unintegrated nuclear DNAs are rapidly loaded with nucleosomal histones (blue). In the case of MLV, NP220 binds to the unintegrated viral DNA and is responsible for attracting histone deacetylases (HDACs), the HUSH complex (consisting of MPP8, TASOR and PPHLN1) and the histone methyltransferase SETDB1. HDACs remove the activation marks of histone acetylation and SETDB1 introduces repressive H3K9me3 marks. MPP8 binds H3K9me3 to strengthen the association with the viral chromatin.

Supplementary information

Supplementary Figure 1

This file contains gel source data - original uncropped source images for EMSA and Western blots.

Reporting Summary

Supplementary Tables

This file contains Supplementary Table 1: Top 100 hits from Genome-wide CRISPR Screen. Top 100 candidate genes identified from CRISPR knockout screen for host factors essential for the silencing of unintegrated MLV DNA. Candidate genes were ranked by analyzing the fold-change in abundance over control and number of enriched sgRNAs per gene using software HiTSelect. This file also contains Supplementary Table 2: qPCR primers. Listed are qPCR primers for MLV total viral DNA (LTR), MLV 2-LTR circular DNA (2LTR), and primers targeting genes MYT1, beta Globin, GAPDH, SETDB1, SETDB2, SUV39H1, SUV39H2, EHMT1, EHMT2, and EZH2.

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Zhu, Y., Wang, G.Z., Cingöz, O. et al. NP220 mediates silencing of unintegrated retroviral DNA. Nature 564, 278–282 (2018). https://doi.org/10.1038/s41586-018-0750-6

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Keywords

  • Unintegrated DNA
  • Silences Proviruses
  • Chromatin Immunoprecipitation (ChIP)
  • Mason-Pfizer Monkey Virus (MPMV)
  • Knockout Cells

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