Unintegrated HIV-1 Dnas Are Loaded with Core and Linker Histones and Transcriptionally Silenced

Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced

Franziska K. Geisa,b,c and Stephen P. Goffa,b,c,1

aDepartment of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032; bDepartment of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032; and cHoward Hughes Medical Institute, Columbia University Medical Center, New York, NY 10032

Contributed by Stephen P. Goff, October 3, 2019 (sent for review July 23, 2019; reviewed by Kate N. Bishop and Melanie Ott) Upon delivery into the nucleus of the host cell, linear double- in permissive cells, the transcriptional silencing is relieved (12, 13). stranded retroviral DNAs are either integrated into the host genome The state of the unintegrated viral DNA, however, only recently is to form the provirus or act as a target of the DNA damage response becoming clarified. The unintegrated DNA of MLV is rapidly and become circularized. Little is known about the chromatinization loaded with core histones and that the histones acquire covalent status of the unintegrated retroviral DNAs of the human immuno- marks characteristic of silent chromatin (14). The silencing of the deficiency virus type 1 (HIV-1). In this study, we used chromatin unintegrated DNA is relieved by histone deacetylase (HDAC) immunoprecipitation to investigate the nature of unintegrated HIV- inhibitors such as trichostatin A, suggesting that histone modifi- 1 DNAs and discovered that core histones, the histone variant H3.3, cations may mediate the silencing (15, 16). An unbiased genome- and H1 linker histones are all deposited onto extrachromosomal wide CRISPR-based knockout screen for genes required for the HIV-1 DNA. We performed a time-course analysis and determined silencing of unintegrated MLV DNAs revealed the involvement that the loading of core and linker histones occurred early after of a large DNA-binding protein (NP220), all 3 subunits of the virus application. H3.3 and H1 linker histones were also found to be eponymous HUSH complex, and the H3K9 trimethyl transferase loaded onto unintegrated DNAs of the Moloney murine leukemia ESET/SETDB1, as well as selected HDACs (17). These various virus. The unintegrated retroviral DNAs are potently silenced, and proteins were shown to bind to the unintegrated MLV DNA and we provide evidence that the suppression of extrachromosomal to be removed from the DNA after integration, coordinately with HIV-1 DNA is histone-related. Unintegrated DNAs were marked by the activation of normal transcription from the provirus. There MICROBIOLOGY posttranslational histone modifications characteristic of transcrip- were indications that different viruses utilized different compo- tionally inactive genes: high levels of H3K9 trimethylation and low nents of the silencing machinery; for example, the HUSH com- levels of H3 acetylation. These findings reveal insights into the plex was more important in silencing unintegrated MLV DNAs nature of unintegrated retroviral DNAs. than unintegrated HIV-1 DNAs. In eukaryotic cells, histones are organized in an octamer nu- unintegrated retroviral DNAs | HIV-1 | histones | transcriptional silencing cleosomal structure, consisting of 2 copies of each the histones H2A, H2B, H3, and H4 (18, 19). Approximately 150 bp of DNA espite extensive research on the human immunodeficiency is wrapped twice around each nucleosome (19). In addition to Dvirus type 1 (HIV-1) life cycle in past years, only the broad canonical core histones, there are also specific core histone vari- outlines of the early steps after infection are known. The detailed ants of H2 and H3 that may contribute to regulation and control events that occur immediately after entry of the retroviral DNA of gene transcription (20). The histone variant H3.3 is a conserved into the nucleus, before the formation of the integrated provirus, protein which shares structurally very high similarity with the are especially unclear. The retroviral life cycle begins with canonical histones H3.1 and H3.2 (21). The function of the H3.3 receptor-mediated fusion of the virion envelope with the host- variant has not been fully elucidated. On one hand, it has been cell membrane and subsequent release of the retroviral capsid into the cytoplasm. The viral RNA genome is reverse transcribed Significance to form a linear double-stranded DNA in a large structure re- sembling the virion core, termed the preintegration complex Retroviruses are characterized by the reverse transcription of (PIC) (1, 2), and this viral DNA complex is delivered into the the viral RNA genome into DNA and the integration of that nucleus by various mechanisms, depending on the virus. The DNA to form the provirus. However, little is known about the PIC of the simple retroviruses, such as the Moloney murine nature of unintegrated HIV-1 DNAs early upon delivery into the leukemia virus (MLV), gains access to the nucleus only after nucleus. Using chromatin immunoprecipitation assays, we found nuclear membrane disassembly during cell mitosis (3, 4). In that both core and H1 linker histones are deposited onto un- contrast, entry of HIV-1 DNA into the nucleus involves active integrated HIV-1 DNAs. We also confirmed transcriptional si- transport through nuclear pores and uses a variety of pore- lencing of unintegrated HIV-1 DNAs and determined the associated Nup proteins (5–8). Once inside the nucleus, the presence of posttranslational histone modifications characteris- linear viral DNA is integrated into the host genome by the viral tic of inactive chromatin. Our results will help to increase the integrase protein (IN), forming the provirus. A portion of the efficiency of expression from nonintegrating HIV-1–based vec- viral DNA that fails to integrate becomes circularized to yield tors and after transient transfections with DNA. circles with either 1 copy of the long terminal repeats (1-LTR circles) (9) or 2 tandem copies of the repeats (2-LTR circles) Author contributions: F.K.G. and S.P.G. designed research; F.K.G. performed research; (10). If proviral formation is prevented either by an IN mutation F.K.G. and S.P.G. analyzed data; and F.K.G. and S.P.G. wrote the paper. or by an IN inhibitor, the circular DNAs tend to accumulate to Reviewers: K.N.B., Francis Crick Institute; and M.O., Gladstone Institutes. higher levels than when integration is allowed (11). The un- The authors declare no competing interest. integrated DNAs—the linear and the circular forms—do not Published under the PNAS license. replicate and gradually disappear as the host cell continues to 1To whom correspondence may be addressed. Email: [email protected]. proliferate. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. It has long been known that the unintegrated retroviral DNAs 1073/pnas.1912638116/-/DCSupplemental. are poorly transcribed and that, after successful proviral formation

www.pnas.org/cgi/doi/10.1073/pnas.1912638116 PNAS Latest Articles | 1of8 Downloaded by guest on September 24, 2021 associated with active transcription (22, 23); on the other hand, it has been suggested as accumulating at silent loci in telomeric or pericentric heterochromatin regions (24–26). Interestingly, it has been shown to play a role in transcriptional silencing of certain retroviral elements in mouse embryonic stem cells (27–29). In addition to the core histones, chromosomal DNA is also condensed by the binding of so-called H1 linker histones. They are required to mediate higher-order chromatin structures. There are 5 somatic subtypes described (H1.1 to H1.5) and, in addition, different H1 variants. In addition to their function in stabilizing nucleosomes, H1 linker histones have been shown to play a role in regulation of gene expression (19, 30, 31). In eukaryotic chromosomes, histones are added either onto daughter DNA strands behind the replication fork or are replaced on DNAs at sites of damage or regions of changing transcriptional regulation. The loading of nucleosomes onto unintegrated retroviral DNA occurs in an extraordinary circumstance, in that the DNA has never previously been associated with histones. How histones are loaded on retroviral DNAs is not yet known. The state of unintegrated HIV-1 DNA and the identities of the histones loaded on the DNA have not been reported. In this study, we investigated the nature of unintegrated HIV-1 DNA. We showed that core histones, the H3 variant H3.3, linker H1 histones, and the posttranslational histone 3 lysine 9 trimethylation (H3K9me3) modification are all rapidly loaded onto unintegrated HIV-1 DNA. Furthermore, we found that H1 linker histones and the histone variant H3.3 were also associated with extrachromosomal Fig. 1. Unintegrated HIV-1 DNAs are potently silenced after nuclear entry. DNAs of MLV. HeLa cells were infected with equal amounts of viral supernatants collected after transfection of 293T cells in parallel with either integrase-deficient Results mutant (IN-D64A) or wild-type IN (IN-wt) DNAs. Cells were monitored 24 h Unintegrated HIV-1 DNAs Are Transcriptionally Silenced Unlike after infection. Data points are the results of independent experiments with Integrated Proviral DNA. We first confirmed the strong silencing of 3 independently produced viral supernatants. The error bars indicate mean ± SD. Statistical significance was determined by unpaired Student’s unintegrated HIV-1 DNAs after infection of HeLa cells. We utilized − − t test. Mock: noninfected control; hi: heat-inactivated viral supernatant as a reporter viruses encoding ZsGreen,basedonthepNL4-3.R .E strain plasmid contamination control. (A) Flow cytometry analyses were con- of HIV-1, pseudotyped with the vesicular stomatitis virus gly- ducted, and representative histograms are shown from IN-D64A and IN-wt coprotein (VSVg) envelope, produced after transfection of 293T infected cells as well as nontransduced control cells (NTD). (B) The percent- cells. We chose to employ a commonly used viral construct age of ZsGreen-positive cells is depicted. *P = 0.032. (C) MFI of ZsGreen- lacking the Vpr protein that would otherwise target various host positive population is illustrated. **P = 0.004. (D) The percentage of proteins for degradation, activate DNA damage responses, and ZsGreen-positive cells and their MFI were multiplied and are represented cause cell cycle arrest (32–34). To better monitor unintegrated relative to IN-wt. (E) Total viral DNA levels were evaluated with qPCR using retroviral DNAs, we utilized an integration-defective mutant (IN- ZsGreen-specific primers. Data were normalized to GAPDH copies. (F) 2-LTR circles were determined by qPCR. Data were normalized to GAPDH levels. D64A), which harbors a single point mutation at the catalytic site of the viral enzyme integrase (35). This mutant allowed normal viral DNA synthesis and nuclear import of the retroviral DNA but PCR (qPCR) utilizing ZsGreen-specific primers to detect total viral prevented integration of the viral DNAs into the host-cell genome. DNA, which includes linear, circular, and (for the IN-wt virus) For comparative studies we used an integration-proficient re- integrated viral DNA. Heat-inactivated virus was used as a porter virus (IN-wt). Producer cells were generated by transfection control for potential plasmid DNA contamination. Importantly, with the 2 viral constructs in parallel, and infections were carried these data exhibited similar levels of total viral DNA of IN- out with equal volumes of virus preparations harvested from the D64A and IN-wt present in the cells (Fig. 1E). We also used producer cells. We performed flow cytometry analyses of reporter qPCR with appropriate primers to assess the levels of 2-LTR gene expression 24 h after infection (representative plots are shown in Fig. 1A). The integrase-defective mutant yielded a sig- circles, specifically reporting the unintegrated viral DNAs nificantly reduced number of ZsGreen-positive cells as compared formed after nuclear entry. As expected, infection with the IN- to the IN-wt (Fig. 1 A and B), and moreover, the mutant resulted D64Avirusresultedinhigherlevelsof2-LTRcirclesarisingas in significantly reduced levels of viral expression compared to IN- a result of the block to integration (Fig. 1F). The results show a wt measured by the mean fluorescence intensity (MFI) of the strong block to expression from the unintegrated HIV-1 DNA ZsGreen-positive population (Fig. 1 A and C). To include con- compared to expression from integrated DNA, as has been seen sideration of both the number of ZsGreen-positive cells and their with MLV (12, 14, 17). MFI after infection, we calculated the product of these 2 readouts. This value for gene expression revealed the strong defect H1 Linker and Core Histones Are Loaded onto HIV-1 Unintegrated in expression from the unintegrated DNAs even more clearly, DNA. We used chromatin immunoprecipitation (ChIP) to di- with an ∼9-fold decrease in the IN-D64A virus as compared to IN- rectly examine the histone content of unintegrated HIV-1 DNAs. wt (Fig. 1D). Notably, we performed an additional readout to HeLa cells were infected with equal amounts of IN-D64A or monitor silencing using viral luciferase reporters. We detected a IN-wt viral supernatants, and at 24 h post infection ChIP was very pronounced silencing of viral expression of IN-D64A– performed followed by qPCR-based analyses of the viral DNAs. infected cells compared to IN-wt in this readout (SI Appendix, ChIPs utilized H1.2- and H1.4-specific antibodies, as members of Fig. S1). To confirm that there was no defect in viral DNA syn- the linker histone family, and H2B- and H3-specific anti- thesis, we isolated DNA and performed a quantitative real-time bodies as representatives of core histones. Two-LTR circle-specific

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1912638116 Geis and Goff Downloaded by guest on September 24, 2021 primers were used for the subsequent qPCR to monitor loading on on the 2-LTR circles occurred rapidly and peaked within 12 h unintegrated virus DNA, and other primers for the cellular after virus application (Fig. 3D). Interestingly, the time course of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase histone loading onto the total viral DNA, based on qPCR with (GAPDH) as an active gene control and the beta globin gene as ZsGreen-specific primers after ChIP, showed more gradually a heterochromatin control. We detected loading of all tested increasing levels, with a peak occurring only at 48 h after virus histone members on the unintegrated HIV-1 DNA of the IN- application (Fig. 3E). qPCR analyses with GAPDH or beta D64A mutant (Fig. 2A) as well as IN-wt (Fig. 2B). H2B and H3 globin-specific primers showed comparable results at all time were present on both GAPDH and beta globin as expected, with points as well as between sample replicates (SI Appendix, Fig. S3 H1.2 and H1.4 antibodies recovering a higher level of beta globin A and B). As control for nonspecific ChIP of DNA, we always DNA compared to GAPDH DNA (SI Appendix, Fig. S2 A and conducted tests with respective isotype control (IgG)-specific B). To test for posttranslational modifications of histones, we antibodies. Rabbit IgG background levels of DNA were uni- used ChIP antibodies to detect H3K9me3, a marker for tran- formly low with primers for 2-LTR circles (Fig. 3D), GAPDH (SI scriptionally inactive chromatin, and acetylated H3 (H3ac), a Appendix, Fig. S3A), or beta globin (SI Appendix, Fig. S3B). marker for transcriptionally active genes. ChIP with H3K9me3 Throughout our studies, however, the rabbit IgG gave signifi- antibodies recovered high levels of unintegrated HIV-1 DNA, cantly elevated background signals when scoring total viral DNA, whereas H3ac antibodies yielded reduced levels as compared to especially at earlier time points after virus application (Fig. 3E). GAPDH (Fig. 2 A and B and SI Appendix, Fig. S2 A and B). As The basis for the background ChIP, seen only with the total viral expected, DNAs recovered with H3ac antibodies were high in DNA primers, is not clear. The background prevented mean- GAPDH and very low in beta globin. In contrast, levels of DNAs ingful readouts of loading on total viral DNA in some cases. associated with H3K9me3 were high in beta globin and low in GAPDH. These data demonstrate that core and H1 histones are The Histone Variant H3.3 Is Loaded onto HIV-1 Unintegrated DNA. The loaded onto unintegrated HIV-1 DNAs and modified with marks H3 histone variant H3.3 has been shown to play a role during associated with silencing. silencing of endogenous retroviral elements (27–29). Therefore, we were motivated to determine whether the H3.3 variant is Histones and H3K9me3 Are Deposited onto HIV-1 Unintegrated DNAs deposited onto unintegrated HIV-1 DNA. To address this Early After Infection. To investigate the timing of histone loading question, cells were exposed to either IN-D64A or IN-wt parti- during the retroviral life cycle, we exposed HeLa cells to IN- cles, and at 24 h post infection ChIP was performed with a H3.3-

D64A particles and after 6 h removed the virus and washed the specific antibody. qPCR analyses of ChIP samples revealed MICROBIOLOGY cells to prevent further infection. We next isolated DNA and substantial levels of unintegrated HIV-1 DNA marked by H3.3 performed qPCR to follow viral DNA synthesis and also con- deposition (Fig. 4A). The experiments also revealed the presence ducted ChIP followed by qPCR at 6, 12, 24, and 48 h post in- of H3.3 histones in the housekeeping GAPDH (Fig. 4B) as well fection (Fig. 3A). The level of total viral DNA, determined by as the heterochromatic beta globin gene (Fig. 4C), although at qPCR with ZsGreen-specific primers, was maximal within 12 h a lower level. after virus application and gradually decreased thereafter (Fig. 3B). qPCR analyses with 2-LTR circle-specific primers revealed Transient H3.3 Knockdown Has Only a Minor Impact on H3.3 Deposition the formation of 2-LTR circles between 6 and 12 h after virus onto Unintegrated HIV-1 DNA. The H3 variant H3.3 is encoded by 2 application (Fig. 3C). To examine the time course of histone genes, H3F3A and H3F3B. The complete knockout of both genes loading, we used H2B- and H3-specific antibodies, assaying the in mice is described to be semilethal (36). To investigate any prototypical core histones, an H1.4-specific antibody, assaying a impact on H3.3 loading onto unintegrated HIV-1 DNA and linker histone family member, as well as a H3K9me3-specific potential changes in viral gene expression, we therefore chose a antibody detecting a major silencing mark. In all cases, the small interfering RNA (siRNA) knockdown (KD) approach, loading of each histone and the formation of the silencing mark transiently knocking down both genes followed by an infection and subsequent analyses after 24 h. siRNA KD of transcripts of either one of the H3F3A and H3F3B genes alone did not prevent H3.3 protein expression, but KD of both together led to an efficient reduction in H3.3 protein levels as assessed by Western blot (Fig. 5 A, Left). Levels were unaffected in nontreated mock cells. These experiments demonstrate that both genes are expressed and that KD of both transcripts is required to efficiently reduce the levels of H3.3 histones. These experiments also demonstrate the specificity of the H3.3-specific antibody utilized throughout our ChIP analyses, since the H3.3 KD led to strong loss of signal detected with H3.3-specific antibody (Fig. 5 A, Left). No loss of signal was detected with the canonical H3-specific antibody (Fig. 5 A, Right). To monitor H3.3 loading after KD, both H3.3 KD and nontargeting siRNA control cells (NT) were exposed to virus, and ChIP experiments were performed at 24 h after infection. Surprisingly, only a minor decrease in levels of DNA recovered Fig. 2. Unintegrated HIV-1 DNA is bound to linker and core histones. IN- with H3.3-specific antibodies was detected in the H3.3 KD cells D64A or IN-wt particles were applied to HeLa cells, and ChIP analyses were compared to NT (Fig. 5 B, Left). Thus, although the levels of conducted after 24 h. Histone-specific antibodies were used for ChIP as in- H3.3 histones in the cells were strongly reduced as assessed by dicated, along with a species-specific isotype control. Mouse IgG (mIgG) Western blot, sufficient H3.3 was still loaded to allow for sub- served as a control for H2B, rabbit IgG (rIgG) for all other antibodies. Data stantial ChIP of the unintegrated HIV-1 DNA. The H3.3 KD are expressed as the percentage of input DNA and are shown as mean ± SD. Data points represent independent experiments with 3 independently produced also led to a small reduction in the levels of unintegrated DNA viral supernatants. (A) ChIP samples from IN-D64A–infected cells were analyzed recovered after ChIP with the canonical H3-specific antibody by qPCR with 2-LTR–specific primers. (B) ChIP samples from IN-wt–infected cells (Fig. 5 B, Right). Interestingly, H3K9me3 levels were slightly in- were analyzed by qPCR with 2-LTR–specific primers. creased in H3.3 KD in contrast to NT control cells (Fig. 5 B, Right).

Geis and Goff PNAS Latest Articles | 3of8 Downloaded by guest on September 24, 2021 Fig. 3. Histone loading onto unintegrated HIV-1 DNAs occurs early after infection. (A) Schematic overview of experimental setup. HeLa cells were infected with IN-D64A virus and washed 3 times at 6 h after virus application, and cells were harvested at indicated time points for qPCR or ChIP with subsequent qPCR. Data points represent 2 independent experiments with 2 independently produced viral supernatants and are shown as mean and range. (B) Cells were pelleted, and DNA was isolated to measure total viral DNA levels by PCR with ZsGreen-specific primers. Data were normalized to GAPDH levels. (C) Cells were harvested, and DNA was isolated to assess 2-LTR circles by qPCR. Data are depicted relative to GAPDH levels. (D) ChIP analyses were conducted using histone- specific antibodies as indicated, along with their respective isotype control (H1.4, H3, H3K9me3: rIgG; H2B: mIgG). qPCR was performed using 2-LTR–specific primers. Data are expressed as percentage of input DNA. (E) qPCRs were performed on ChIP samples with ZsGreen-specific primers to detect total viral DNA. Data are shown as percentage of input DNA.

Of note, qPCR analyses after ChIP with GAPDH- or beta with both IN-D64A and IN-wt virus. Combining the readouts globin-specific primers exhibited no changes in ChIP with H3 of the ZsGreen-positive cells and the MFI, by multiplying both or H3K9me3 antibodies (SI Appendix,Fig.S4A and B, Left) values, revealed only a minor increase in viral gene expression and only a minor decrease in H3.3 antibodies (SI Appendix,Fig. for H3.3 KD cells treated with IN-D64A compared to NT S4 A and B, Right). control cells (Fig. 5E). To address a possible correlation of H3.3 KD and viral gene expression, we exposed siRNA-treated cells to either IN-D64A Unintegrated DNAs of MLV, like Those of HIV-1, Are Loaded with H1 or IN-wt particles of indicated volumes of viral supernatants and Linker and H3.3 Histones. In addition to test histone loading on monitored expression. Flow cytometry analyses were conducted HIV-1–based reporter viruses, we performed analyses with MLV, 24 h after virus application. H3.3 KD cells exhibited a very another member of the retrovirus family. Our group has previously modest increase in ZsGreen-positive cells (Fig. 5C)andinMFI shown that core histones are loaded onto unintegrated MLV DNA levels (Fig. 5D) as compared to control cells after infection early after nuclear entry (14). We utilized an MLV-based reporter

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1912638116 Geis and Goff Downloaded by guest on September 24, 2021 both MLV and HIV-1. The significance of this variant for viral DNA processing or expression remains unclear, but may contribute to the limited expression of these DNAs. In addition, and unexpectedly, we detected loading of the linker histone H1 on extrachromosomal HIV-1 and also MLV DNAs. These histones are traditionally associated with higher-order condensation of chromatin and thus may induce a compaction of the DNAs. Linker histones are also described in the literature to play a role in gene regulation, and numerous posttranslational modifications are known, but their function is poorly understood (30). It would be of interest to analyze potential correlations between Fig. 4. The histone variant H3.3 is loaded onto unintegrated HIV-1 DNA. linker histone H1 and regulation of retroviral gene expression. Equal amounts of viral supernatants of IN-D64A or IN-wt particles were It has been previously shown that the unintegrated DNAs of applied to HeLa cells, and ChIP analyses with H3.3-specific antibodies and – rabbit IgG were performed after 24 h. Data are depicted as percentage of both HIV-1 and MLV are heavily silenced (12 14, 17). Here we input DNA and are shown as mean ± SD; data points represent independent provide confirmation that the silencing is pronounced; delivery experiments with 3 independently produced viral supernatants. (A) qPCR of a ZsGreen reporter gene by IN-defective HIV-1–based vector reactions were performed on ChIP samples with 2-LTR–specific primers. (B) induced significantly fewer fluorescence-positive cells with a qPCRs of ChIP samples were performed with GAPDH-specific primers for lower MFI compared to those induced by the IN-wt vector, even housekeeping control. (C) ChIP samples were analyzed by qPCR using beta though the total viral DNA levels were comparable. We detected globin-specific primers as a heterochromatin control. high levels of the posttranslational histone modification H3K9me3, a silencing mark, and low levels of H3ac modification, an activation mark. Both modifications correlate with the virus harboring an IN-inactive mutation D184A and encoding observed silencing phenotype of unintegrated HIV-1 DNAs. GFP. We infected HeLa cells and performed ChIP experiments We showed by time-course analyses that histone deposition after 24 h. Both the circular unintegrated MLV DNAs, scored and H3K9me3 modification onto unintegrated HIV-1 DNA – A using 2-LTR specific primers (Fig. 6 ), and the total unintegrated appears rapidly after nuclear entry. Notably, the kinetics of MLV DNA, scored using GFP-specific primers (Fig. 6B), were loading onto circles, judged by ChIP and subsequently qPCR decorated with linker histones, shown by H1.4 deposition, as well utilizing 2-LTR circle-specific primers, exhibited a peak at 12 h MICROBIOLOGY as with the H3.3 variant. qPCR analyses of the DNAs after ChIP after infection, while the loading on total DNA, utilizing primers with primers specific for GAPDH (Fig. 6C)orbetaglobin(Fig. for detecting total virus, showed a more gradual increase. It 6D) showed expected levels of H1.4 and H3.3. Thus, like HIV-1 should be noted that the readout of total viral DNA detects DNAs, unintegrated MLV DNAs are also loaded with both H1 many species, including DNA intermediates in the cytoplasm, linker and H3.3 variant histones. DNAs in PICs that have not uncoated, as well as nuclear DNAs. Discussion In contrast, the readout of circular DNA only detects nuclear forms that are likely fully uncoated and therefore are more ac- The suppression of incoming DNA by transcriptional silencing is cessible both for the host DNA damage repair machinery and for a major aspect of cellular innate immunity, constituting a block histone loading. These aspects of the assay likely account for the to the initiation of infection by invading viruses. The system is difference in the timing of loading, as measured by the recovered known to act against many incoming viruses, including MLV (17), DNA after ChIP relative to the input DNA. and many DNA viruses, such as herpes viruses (37, 38), adeno- In all our ChIP experiments, we always performed controls viruses (39), and adeno-associated viruses (40). In all these cases with nonspecific isotype-matched Ig to determine the baseline the mechanism involves the loading of histones onto the viral levels of DNA recovered nonspecifically in the immunoprecipi- DNA and the posttranslational modification of these histones to tates. These background levels of DNA are almost always ex- induce silencing and regulate gene expression. The importance of tremely low. We experienced difficulties, however, with high the antiviral system is highlighted by the fact that many viruses backgrounds throughout our studies when using rabbit IgG an- have evolved genes that function to inactivate the silencing ma- tibodies for ChIP and qPCR primers detecting total HIV-1 viral chinery and allow viral expression, such as Vpr of HIV-1 and Vpx DNA. In some cases, these high backgrounds prevented mean- of simian immunodeficiency virus type 1 (SIV-1) (41–44). The ingful readouts of histone loading on total HIV-1 DNAs. We system is also active in suppression of expression of nonviral found these high backgrounds only with HIV-1, not MLV; only DNAs introduced by many transfection methods, and inhibition of with total viral DNA and not 2-LTR; and only with rabbit IgG the silencing machinery can result in dramatic enhancement of the and not mouse IgG. The data suggest that there may be some level of expression of such DNAs (45, 46). component of the HIV-1 PICs that binds rabbit IgG non- In this study, we investigated the nature of the chromatin specifically. A possible candidate for this is the host protein formed on unintegrated HIV-1 DNAs, its histone composition, TRIM21, a cytosolic Fc receptor capable of binding IgGs (47) and the time course of histone loading. We made use of IN- and known to be specifically packaged into HIV-1 virions (48). deficient virus particles to assure analysis of purely unintegrated Investigators using ChIP to probe HIV-1 PICs should be alerted DNAs. The circumstances of histone loading on these DNAs are to this potentially confounding IgG-binding activity. Importantly, unusual, in that nucleosome formation needs to take place on the isotype controls for the other target DNAs such as 2-LTR “virgin” DNA, which has never previously been associated with circles, GAPDH, or beta globin DNAs uniformly gave the usual histones. As we previously have reported with MLV (14), we very low, negligible backgrounds. detected rapid loading of core histones, such as H3 and H2B, on We were interested in exploring the potential role of the H3.3 unintegrated HIV-1 DNA. HIV-1 and MLV enter the nucleus variant histone in the chromatinization of the viral DNAs and their through very different mechanisms: MLV requires nuclear transcriptional silencing. Because the complete knockout in the membrane disassembly, while HIV-1 engages with nuclear pore mouse germ line is semilethal (36), we used siRNA-mediated proteins. Thus our studies show that histone loading onto the knockdowns to specifically reduce H3.3 expression and monitored unintegrated retroviral DNAs occurs independently of the route the H3.3 loading onto viral DNA. It proved almost impossible to of nuclear entry. Using specific antisera, we also demonstrate prevent H3.3 loading onto unintegrated HIV-1 DNA, even though loading of the histone variant H3.3 on unintegrated DNAs of the overall levels of the histone variant could be dramatically

Geis and Goff PNAS Latest Articles | 5of8 Downloaded by guest on September 24, 2021 Fig. 5. Transient H3.3 KD has a minor impact on H3.3 deposition onto unintegrated HIV-1 DNA and its expression. (A) Cells were transfected twice on 2 consecutive days either with siRNAs against H3F3A or H3F3B or with siRNAs against both H3F3A and H3F3B. Mock cells served as a nontreated control. Cells were harvested and lysed 24 h after the second siRNA transfection. Western blot was performed using an H3.3-specific antibody (Left) or an H3-specific antibody (Right). GAPDH-specific antibody was used as a housekeeping control. (B) HeLa cells were transfected twice on 2 consecutive days with siRNAs against H3F3A and H3F3B, both encoding H3.3, and with a nontargeting siRNA control (NT). H3.3 KD or NT cells were infected 24 h after the second siRNA transfection with IN-D64A virus. Data are depicted as mean ± SD, and data points represent independent experiments with at least 3 independently produced viral supernatants. ChIP analyses with indicated histone-specific antibodies and their isotype control were performed 24 h after virus application. qPCR data for 2-LTR–specific primers are shown as percentage of input DNA. (C)H3.3KD(H3F3A+H3F3B) or NT cells were infected 24 h after the second siRNA transfection with various volumes of viral supernatants of IN-D64A or IN-wt virus as indicated. Data are shown as mean and range, and data points represent independent experiments with independently produced viral supernatants. Flow cytometry analyses were conducted, and the percentage of ZsGreen-positive cells is shown. Data are depicted as mean and range from 2 to 3 independently produced viral supernatants as indicated. (D) MFI from the same experiment is shown. (E)The product of the percentage of ZsGreen-positive cells and their MFI is presented relative to control NT cells for each setting.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1912638116 Geis and Goff Downloaded by guest on September 24, 2021 of loading. These events, occurring very early after exposure to virus, can have a major impact on the outcome of the infection and the efficiency of virus transmission. Moreover, the potent silencing of unintegrated HIV-1 DNAs constitutes a major limita- tion for the use of nonintegrating retroviral vectors for gene therapy applications. Modulation of posttranslational histone modifications through specific inhibitors has been shown to improve gene expression in transient DNA transfection applications (49, 50), and controlling histone-based silencing may similarly improve the utility of nonintegrating viral vectors. Materials and Methods Retrovirus Reporter Plasmids. HIV-1–based reporter virus construct pNL4- − − 3.ZsGreen.R .E , described previously (51), was used throughout the study and is here referred to as IN-wt. We generated the IN-deficient mutant − − pNL4-3.ZsGreen.R .E (IND64A), which is here named IN-D64A, by trans- − − ferring the XhoI–NotI fragment from pNL4-3.Luc.R .E (IND64A) (17). The mutant harbors a mutation at the catalytic site of the retroviral enzyme integrase and allows nuclear entry but prevents integration (35). The Moloney MLV-based expression constructs pCMV-intron.IND184A (Gag-Pol from NB-tropic MLV) and pNCA-GFP (MLV expressing GFP) were as previously published (14). All reporter virus vectors had a replication-defective single-round design and were pseudotyped with the VSV G envelope encoded by pMD2.G (a gift from Didier Trono, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland; Addgene plasmid, 12259), referred to as VSVg here.

Cells and Cultivation. Human HeLa cell line (American Type Culture Collec- tion, CCL-2) and human Lenti-X 293T cell line (Clontech, 632180), were cul- MICROBIOLOGY tured in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% Fig. 6. Histone variant H3.3 and linker histones are deposited onto un- heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL integrated MLV DNAs. HeLa cells were infected with MLV integrase-deficient streptomycin. mutant IN-D184A virus encoding GFP, and ChIP analyses were carried out with histone-specific antibodies and their isotype control 24 h after virus Retroviral Particle Production and Infection. One day before transfection, 5 × application. Data shown are percentage of input DNA, as mean ± SD. Data 106 293T cells per 10-cm dish were seeded. Retroviral particle production was points represent independent experiments with 3 independently produced achieved by the calcium phosphate precipitation method using CaCl and 2x viral supernatants. (A) ChIP samples were used to perform qPCR with 2-LTR– 2 Hepes-buffered saline (pH 7.08). Culture medium was supplemented with specific primers. (B) qPCR reactions were performed on ChIP samples with 10 mM Hepes during particle production. To package HIV-1–based reporter GFP-specific primers to detect total viral DNA. (C) qPCR reactions were virus, 10 μg IN-wt or IN-D64A plasmid DNA was cotransfected with 2 μg VSVg performed with GAPDH-specific primers as a housekeeping control. (D) qPCR DNA in a 10-cm dish. To produce Moloney MLV-based reporter virus, 10 μg reactions were performed on ChIP samples with beta globin-specific primers pNCA.GFP plasmid was transfected along with 15 μg pCMV-intron (encoding as a heterochromatin control. NB-tropic MLV Gag-Pol) and 2 μg VSVg per 10-cm dish. Cells were washed ∼5h after transfection. Viral supernatants were harvested 36 and 48 h after transfection, filtered through 0.45-μm pore-size filters and concentrated reduced as assessed by Western blots. The residual pool of H3.3 (100×) by ultracentrifugation (2 h, 25,000 rpm, 4 °C). Viral pellets were in KD cells apparently provided adequate supplies for loading resuspended in culture medium and stored in aliquots at −80 °C until further on the incoming virus. It should be noted that the quantities of usage. HeLa cells were infected with viral supernatants and incubated for at viral DNA are tiny in comparison to the host genomic DNA: at least 5 h if not indicated otherwise. The concentrated IN-wt virus prepa- multiplicities of infection of ∼1, they represent roughly one or a rations typically exhibited titers on HeLa cells of ∼2.5 × 108 transducing few one-hundred thousandths of the host DNA by weight. There units per milliliter. IN-wt and IN-D64A viruses were simultaneously pro- was only a very slight reduction in the levels of H3.3 loaded and duced, and equal amounts of supernatants were applied to cells within an very little change in the levels of expression from the viral reporter experiment for comparative studies. We typically utilized a multiplicity of gene. It thus remains unclear if complete loss of H3.3 would have infection of ≤0.5, which leads to ∼30 to 50% fluorescent-positive cells. a larger effect on silencing. The state of condensation of the unintegrated retroviral DNAs Flow Cytometry. Cells were harvested at respective time points after infection with 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA) and resuspended may well contribute to their transcriptional silencing. In this in phosphate-buffered saline (PBS) supplemented with 3% heat-inactivated regard, the presence of the H1 linker histones on the viral DNA FBS. The BD LSRII flow cytometer (BD Biosciences) was used, and analyses is intriguing. It may be that the H1 organizes the viral chromatin were conducted using FlowJo software. Data were first gated for viable into a highly condensed state and thereby reduces access to the cells through forward scatter height and side scatter height. ZsGreen- DNA by the transcriptional machinery. Analysis of the density positive cells were then scored by gating from the previously defined via- and spacing of nucleosomes on the PICs, as assessed by micro- ble population. MFI was determined based on the gated ZsGreen-positive coccal nuclease digestion or assay for transposase-accessible population. chromatin-sequencing (ATAC-seq) methods, could be in- 6 formative. Tests for the role of H1 histones in determining such ChIP. One day before infection, 2 × 10 HeLa cells per 10-cm dish were structures might be of great importance. Another important seeded. Before infection, viral supernatants were pretreated with 5 U/mL question for the future is the identification of the host machinery DNase I (Promega, M6101) for 1 h at 37 °C to remove any residual plasmid DNA. Media during infection were supplemented with 8 μg/mL Polybrene. involved in histone loading. Manipulation of specific histone Cells were rinsed twice with 1 × PBS and cross-linked with 1% formalde- chaperones will reveal the major loaders and could determine hyde for 10 min at 37 °C, quenched in 0.125 M glycine for 5 min, and lysed in the importance of histone variants such as H3.3. 700 μL of ChIP lysis buffer (50 mM Tris·HCl, pH 8.0, 1% sodium dodecyl sul- To summarize, we described here the chromatinization of fate, 10 mM EDTA). Cell lysates were sonicated, immunoprecipitated, and unintegrated HIV-1 DNA, its histone composition, and the timing reverse–cross-linked. After DNA isolation, qPCR was conducted with ChIP

Geis and Goff PNAS Latest Articles | 7of8 Downloaded by guest on September 24, 2021 samples. Extended information for ChIP protocol is provided in SI Appendix, twice on 2 consecutive days, cells were seeded for infection at the end of Supplementary Materials and Methods. the day of the second siRNA transfection, and infection was accomplished 24 h later. Cells were harvested 24 h after infection for ChIP or 30 h after Detection of 2-LTR Circles and Total Viral DNA. HeLa cells were pelleted at infection for flow cytometry analysis, and cells were pelleted for subsequent indicated time points, and DNA was isolated with the QIAamp DNA blood Western blot after the last harvest of cells to confirm KD during all experi- mini kit following the manufacturer’s instructions (Qiagen). Approximately mental procedures. Detailed Western blot protocol can be found in SI Ap- 50 to 100 ng DNA per sample was used for subsequent qPCR analyses. The pendix, Supplementary Materials and Methods. cycle threshold (Ct) values were normalized to endogenous GAPDH levels ΔCt based on the 2 method. Extended information for qPCR detection of viral Statistical Analysis. Data were shown as mean ± SD or mean and range as DNAs is provided in SI Appendix, Supplementary Materials and Methods. indicated. We used unpaired t test for comparison of 2 groups, and in the case of high variances between the two groups, Welch’s correction was siRNA Transfection and Western Blot. The following siRNAs were purchased applied. P values ≤ 0.05 were considered significant (*) and ≤ 0.01 very from Dharmacon with ON-TARGET plus SMART pool design: H3F3A (L-011684- significant (**). 01-0005); H3F3B (L-012051-00-0005); and nontargeting pool (D-001810-10-20). × 5 For siRNA transfection, 4 10 HeLa cells per 10-cm dish were seeded, ACKNOWLEDGMENTS. S.P.G. is an HHMI investigator. This study was and the next day, transfection was performed with 300 pmol siRNA per supported in part by a grant from the NIH (CA 30488). F.K.G. was supported dish and Lipofectamine RNAiMAX (Life Technologies, 137785000) for at least by the Deutsche Forschunsgemeinschaft (German Research Foundation) 5 h and according to the manufacturer’s advice. Transfection was carried out Grant GE 3106/1-1.

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