HIV-1 Vpr induces cell cycle arrest and enhances viral gene expression by depleting CCDC137

The HIV-1 Vpr accessory protein induces ubiquitin/proteasome-dependent degradation of many cellular proteins by recruiting them to a cullin4A-DDB1-DCAF1 complex. In so doing, Vpr enhances HIV-1 gene expression and induces (G2/M) cell cycle arrest. However, the identities of Vpr target proteins through which these biological effects are exerted are unknown. We show that a chromosome periphery protein, CCDC137/cPERP-B, is targeted for depletion by HIV-1 Vpr, in a cullin4A-DDB1-DCAF1 dependent manner. CCDC137 depletion caused G2/M cellcycle arrest, while Vpr-resistant CCDC137 mutants conferred resistance to Vpr-induced G2/M arrest. CCDC137 depletion also recapitulated the ability of Vpr to enhance HIV-1 gene expression, particularly in macrophages. Our findings indicate that Vpr promotes cell-cycle arrest and HIV-1 gene expression through depletion of CCDC137.


Introduction
Human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIVs) encode several accessory proteins; Vpr, Vpx, Vif, Nef, and Vpu. While accessory proteins are often dispensable for replication in immortalized cell lines, they are important in a physiological context and typically act by removing or displacing molecules that are deleterious to virus replication. Among the HIV-1 accessory proteins, the function of~14 kDa Viral Protein R (Vpr) remains the most enigmatic. Replication deficits of inconsistent magnitude are evident in HIV-1 mutants lacking Vpr, particularly in primary macrophages (Balliet et al., 1994;Connor et al., 1995;Fouchier et al., 1998), while deletion of Vpr from SIVmac modestly attenuates pathogenesis (Hoch et al., 1995;Gibbs et al., 1995).

Identification of CCDC137 as a Vpr target protein
To identify HIV-1 Vpr target proteins, we used a proximity-dependent method (Roux et al., 2012) in which a biotin-ligase, BirA(R118G) fused an HIV-1 NL4-3 Vpr bait was expressed in proteasome inhibitor treated cells. Biotinylated proteins were enriched using streptavidin magnetic beads, identified using mass spectroscopy and the biotinylated proteome in BirA(R118G)-Vpr, and BirA(R118G) expressing cells was compared. Multiple nuclear proteins were biotinylated in BirA(R118G)-Vpr, but not BirA(R118G) expressing, cells ( Based on the fact that Ki-67 was the top hit in our proximity biotinylation experiments but did not itself appear to be responsible for the Vpr-induced eLife digest Like all viruses, the human immunodeficiency virus 1 (HIV-1) cannot replicate on its own; to multiply, it needs to exploit the molecular machinery of a cell. A set of HIV-1 proteins is vital in this hijacking process, and they are required for the virus to make more of itself. However, HIV-1 also carries accessory proteins that are not absolutely necessary for the replication process, but which boost the growth of the virus by deactivating the defences of the infected cells. Amongst these proteins, the role of Viral Protein R (Vpr for short) has been particularly enigmatic.
Previous experiments have shown that, in infected cells, Vpr is linked to several biological processes: it tags for destruction a large number of proteins, it causes the cells to stop dividing, and it encourages them to express the genetic information of the virus. How these different processes are connected and triggered by Vpr is still unknown. It particular, it remains unclear which protein is responsible for these changes when it is destroyed by Vpr.
To investigate, Zhang and Bieniasz conducted a series of experiments to spot the proteins that interact with Vpr in human cells. This screening process highlighted a protein known as CCDC137, which is depleted in cells infected by HIV-1.
To investigate the role of CCDC137, Zhang and Bieniasz decreased the levels of the protein in human cells. This stopped the cells from dividing, just like during HIV-1 infection. Destroying CCDC137 also mimicked the effects of Vpr on HIV-1 gene expression, increasing the levels of virus proteins in infected cells. Finally, Zhang and Bieniasz made a mutant version of CCDC137 that Vpr could not destroy. When infected cells carried this mutant protein, they kept on dividing as normal. Taken together, these results suggest that Vpr works by triggering the destruction of the CCDC137 protein. Overall, this work represents the first step to understand the role of CCDC137 in both infected and healthy cells.
cell cycle arrest, we hypothesized that a Ki-67-proximal or interacting protein might represent the genuine target of Vpr. Ki-67 recruits a group of proteins termed 'chromosome periphery proteins' (cPERPs), that localize within the nucleus, primarily the nucleolus, during interphase but are relocalized to chromosome peripheries during mitosis (Booth et al., 2014;Ohta et al., 2010). Therefore, we next conducted a focused screen of candidate target proteins that were either prominent hits in the BirA(R118G)-Vpr screen (Supplementary file 1), were nucleolar or nuclear proteins, members of the cPERP group, and/or were reported to bind Ki-67. Of numerous candidates tested in transient co-transfection/western blot assays, Vpr only induced the depletion of cPERP-B, also termed CCDC137 (Ohta et al., 2010;Figure 1-figure supplement 2A-C). To assess the potency with which Vpr could induce depletion of CCDC137, we cotransfected 293 T cells with various amounts of CCDC137 and HIV-1 Vpr expression plasmids. Both CCDC137 and Vpr proteins were tagged with the same epitope, a single copy of an HA tag, to assess their relative steady-state levels. This analysis revealed that co-expression of a barely detectable amount of Vpr resulted in the removal of much larger quantities of CCDC137, underscoring the potency with which Vpr induced CCDC137 depletion ( Figure 1A,B, Figure 1-figure supplement 2D). Treatment of cells with MG132 abolished the ability of Vpr to induce CCDC137 depletion, suggesting that Vpr triggered proteasomedependent CCDC137 degradation ( Figure 1C). Furthermore, when DCAF1, an essential component of the CRL4 complex, was depleted by lentiviral vector-mediated RNA interference, the depletion of CCDC137 was abrogated ( Figure 1D).

Derivation of Vpr-resistant CCDC137 mutants
We set out to mapped the degrons through which CCDC137 is depleted by Vpr. First, CCDC137 was expressed as N-terminal (residues 1-154) and C-terminal (residues 155-289) fragments. Transfection experiments revealed that the isolated N-terminal but not the C-terminal portion was depleted by Vpr ( Figure 2-figure supplement 1A). Alanine scanning mutagenesis in the context of full length CCDC137, whereby blocks of 5 residues were mutated throughout the N-terminal CCDC137 portion revealed that CCDC137 residues 61 to 75 were important for Vpr-induced depletion ( Figure 2A, Figure 2-figure supplement 1B). Additionally, alanine substitutions of an LxxLL motif (positions 228-232) through which CCDC137 binds nuclear receptors (Youn et al., 2018), also reduced Vpr-induced CCDC137 depletion (L/A, mutation, Figure 2A). Combined substitution of  CCDC137 residues 61-65 or 66-70, coupled with the L/A (228-232) mutation caused CCDC137 to be substantially Vpr-resistant ( Figure 2A). As previously reported, CCDC137 formed foci in the nucleus that colocalized with fibrillarin, a nucleolar marker, during interphase (Ohta et al., 2010; Figure 2B) and this property was unaffected by the aforementioned residue 61 to 65, 66 to 70, or the L/A (228-232) Vpr resistance-inducing substitutions ( Figure 2C).

Physical association of CCDC137 and Vpr
To determine whether Vpr and CCDC137 were physically associated, we co-expressed a glutathione S-transferase (GST)-Vpr fusion protein with CCDC137 in 293 T cells. Wild-type CCDC137, but not a Vpr-resistant CCDC137 mutant (L/A 66-70), could be co-precipitated with GST-Vpr ( Figure 3A) when coexpressed in 293 T cells. Conversely, a mutant GST-Vpr (Q65R) that did not cause CCDC137 depletion (Figure 3-figure supplement 1) did not co-precipitate CCDC137 ( Figure 3A). Similarly, V5-tagged CCDC137 could be co-immunoprecipitated with HA-tagged Vpr, but other control proteins, including Ki-67-interacting protein (MKI67IP) and nucleolar-localized ribosomal RNA processing protein 1 (RRP1), did not coimmunoprecipitate with Vpr ( Figure 3B). When the biotinylation proximity assay (Roux et al., 2012) was coupled to Western blot analysis, CCDC137 was found to be biotinylated by Vpr-BirA(R118G) but not BirA(R118G) despite our inability to detect biotinylated CCDC137 in the initial mass spectroscopic screens ( Figure 3C).

Endogenous CCDC137 depletion in HIV-1 infected cells
We infected 293T or U2OS cells with a minimal version of HIV-1 (termed V1) in which Gag, Pol, Vif, Vpu and Env carry deletions or nonsense mutations. In V1 infected cells, Tat, Rev, HA-tagged Vpr and GFP are expressed, all driven by native HIV-1 LTR sequences, and infected cells can be identified by flow cytometry or microscopy (Figure 4-figure supplement 1). Infection with V1 lacking Vpr (V1/d-Vpr) had no effect on endogenous CCDC137 levels, while infection with V1/HA-Vpr, encoding Vpr from one of several HIV-1 laboratory adapted or primary transmitted founder strains, caused profound CCDC137 depletion ( Figure 4A). HIV-2 Vpr induced partial CCDC137 depletion while SIV MAC Vpr did not affect CCDC137 levels ( Figure 4A), consistent with their abilities, or lack thereof in the case of SIVmac, to induce G2M arrest (Figure 4-figure supplement 2). Immunofluorescent staining of endogenous CCDC137 in 293 T cells, or ectopically expressed V5-tagged CCDC137 in U2OS cells, showed that CCDC137 was diminished to nearly undetectable levels in V1/

CCDC137 depletion causes G2/M cell cycle arrest and a DNA damage response
To ascertain whether CCDC137 depletion might be responsible for Vpr-induced cell cycle arrest, we next asked whether depletion of CCDC137 per se could induce G2/M cell cycle arrest. Lentiviral constructs expressing CAS9 and CCDC137-targeting CRISPR guide RNAs efficiently generated CCDC137 knockout alleles, but most transduced cells died and none of the numerous surviving cell clones that were analyzed contained frameshifting indels in both copies of CCDC137, suggesting that CCDC137 is essential for proliferating cell viability. Two CCDC137-targeting, lentiviral shRNA vectors encoding a puromycin N-acetyl-transferase caused effective short term CCDC137 depletion after puromycin selection of 293 T cells, with CCDC137/shRNAII causing more profound depletion than CCDC137/shRNAI ( Figure 5A). Notably, CCDC137/shRNA transduced cells accumulated in G2/M ( Figure 5B) as revealed by propidium iodide (PI) staining and the extent of CCDC137 depletion and G2/M accumulation were correlated ( Figure 5A,B). A CCDC137 cDNA construct lacking the 3'UTR targeted by CCDC137/shRNAII substantially rescued G2/M arrest ( Figure 5B). As an alternative way to assess the effect of CCDC137 knockdown on cell cycle, we utilized a Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) construct (Sakaue-Sawano et al., 2008), which couples the cell-cycle regulated proteins with fluorescent proteins. G2/M accumulation was also evident upon CCDC137 depletion in several U2OS cell clones expressing mClover or mKusabira-Orange2 (mKO2) fluorescent proteins fused to geminin (1-110 aa) that are depleted during G1 but present during G2/M ( Figure 5C, Figure 5-figure supplement 1). Accordingly, live cell imaging of U2OS/ mClover-hGeminin (1-110 aa) cells revealed fluctuating fluorescence that disappeared upon cell division, while CCDC137 depleted cells did not divide and retained mClover-hGeminin fluorescence, indicating G2/M growth arrest until apparent cell death ( Figure 5-video 1). HIV-1 and HIV-2 Vpr are able to activate the DNA damage response (DDR) which activates response pathways through ATM/ATR and Chk1/2 kinases, leading to cell cycle arrest (Roshal et al., 2003;Zimmerman et al., 2004;Fregoso and Emerman, 2016). As part of the Vpr-induced DNA damage response, histone H2A variant H2AX, a marker for DNA damage, undergoes phosphorylation at Ser 139 (g-H2AX) and forms nuclei foci. CCDC137 depletion using shRNA also caused accumulation of nuclear foci of g-H2AX ( Figure 5D), mimicking the reported Vpr-induced DDR. Importantly, the CCDC137 depletioninduced DDR was rescued by expression of a shRNA-resistant CCDC137 cDNA ( Figure 5D).

Vpr-resistant CCDC137 mutants abrogate Vpr-induced G2/M cell cycle arrest
If Vpr induces cell-cycle arrest through depletion of CCDC137, we reasoned that Vpr-induced cellcycle arrest should be alleviated by overexpression of the Vpr-resistant CCDC137 mutants. U2OS cells containing doxyxcycline inducible CCDC137 expression constructs were treated with doxycycline to induce the expression of CCDC137 and then infected with V1/HA-Vpr or V1/d-Vpr at an MOI of 2 for analysis of protein levels ( Figure 6A), or an MOI of 0.5 for analysis of DNA content ( Figure 6B). Wild-type CCDC137 was depleted following V1/HA-Vpr infection while mutant CCDC137 (L/A 66-70) largely resisted Vpr-induced depletion ( Figure 6A). Crucially, overexpression of WT CCDC137 partly ameliorated the G2/M arrest effect of Vpr, while expression of the Vpr-resistant CCDC137 (L/A 66-70) mutant conferred nearly complete resistance to Vpr-induced G2/M arrest ( Figure 6B,C), suggesting that depletion of CCDC137 is necessary for G2/M cell cycle arrest induction by Vpr.

Enhancement of HIV-1 gene expression by CCDC137 depletion
Prior work has shown that Vpr enhances HIV-1 HIV-1 gene expression in a variety of cell types (Connor et al., 1995;Goh et al., 1998;Gummuluru and Emerman, 1999). Both of these phenotypes have also been associated with G2/M arrest properties of Vpr. We therefore assessed the effects of Vpr expression or CCDC137 depletion on HIV-1 gene expression in various cell types.

CCDC137 depletion increases HIV-1 gene expression in primary cells
While Vpr expression caused CCDC137 depletion and increased HIV-1 gene expression in U2OS cells, these cells are not natural targets of HIV-1. Therefore, we next examined the effects of Vpr on CCDC137 and HIV-1 gene expression in primary cells.
Immunofluorescence assays in V1/HA-Vpr and V1/d-Vpr infected primary macrophages showed that Vpr effectively depleted CCDC137 therein while the Q65R mutant Vpr did not ( Figure 8A, Figure 8-figure supplement 1). Infection of primary CD4+ T-cells with V1/HA-Vpr and V1/d-Vpr revealed that the presence of Vpr resulted in~2 fold higher levels of GFP expression than did in its absence ( Figure 8B,C). The effect of Vpr on HIV-1 gene expression was more pronounced in primary macrophages, where V1/HA-Vpr but not V1/HA-Vpr(Q65R) resulted in higher levels of GFP expression than did infection with V1/d-Vpr, even though marked donor-to-donor variation was evident ( Figure 8D,E, Figure 8-figure supplement 1, Figure 8-videos 1, 2). Several different Vpr proteins from primary HIV-1 strains caused increased HIV-1 gene expression in macrophages ( Figure 8F) and similar Vpr-induced enhancement of HIV-1 gene expression was evident in macrophages infected with a full-length reporter virus (HIV-1 NHG , Figure 8-figure supplement 2), as previously reported (Connor et al., 1995). Notably, the Vpr-induced increase in GFP levels in V1 infected macrophages was accompanied by elevated HIV-1 RNA levels, as assessed by in-situ hybridization ( Figure 8G,H) indicating that Vpr enhances viral gene expression primarily by enhancing RNA synthesis or increasing RNA stability.
To enable Vpr-independent CCDC137 depletion in infected primary cells, while simultaneously measuring HIV-1 gene expression, we constructed a derivative of V1/d-Vpr carrying an shRNA expression cassette (V1/sh, Figure 9 . This enhancing effect of CCDC137 depletion on HIV-1 gene expression was also evident when HIV-1 RNA levels were assessed by in-situ hybridization assays using probes targeting GFP ( Figure 9D,E). Similarly, qRT-PCR ( Figure 9F) assays of GFP and Gag mRNA levels in multiple donors, indicated that CCDC137 depletion recapitulated the effect of Vpr on HIV-1 transcription or RNA stability. Overall, in three different cell types, shRNA-driven CCDC137 depletion had similar enhancing effects on HIV-1 gene expression as did Vpr expression.

Discussion
A persistent paradox in the study of Vpr is that Vpr-defective viruses are often selected in long term replication experiments or in chronically infected cells (Jowett et al., 1995;Rogel et al., 1995). Presumably therefore, Vpr is deleterious to HIV-1 replication in these contexts, despite its positive effects on HIV-1 gene expression. This effect seems likely to be explained the interplay of infected cell life span and burst size (Goh et al., 1998). In cell culture, the longevity of an infected cell plays a crucial role in determining burst size, and thus the effect of Vpr in curtailing the lifespan of an infected cell is expected to confer a competitive disadvantage. Conversely, the life span of infected T-cells in vivo is likely limited by factors other than Vpr and even a modest Vpr-induced enhancement in viral gene expression should confer advantage. Macrophages are not cycling cells, and lack ATR, Rad17 and Chk1 (Zimmerman et al., 2006). Thus, G2/M arrest and DDR induction by Vpr are likely not relevant therein. We confirmed herein that the Vpr-induced enhancement of HIV-1 gene expression is particularly evident in macrophages (Connor et al., 1995). We surmise that the key  While this work was in progress, Greenwood et al reported the effects of HIV-1 infection on the cellular proteome and found that many proteins were depleted from HIV-1 and HIV-2 infected cells, in a Vpr-dependent manner (Greenwood et al., 2019). Consistent with our findings, CCDC137 was among the proteins found to be depleted in HIV-1 and HIV-2 infected cells. While CCDC137 was not investigated further, Greenwood et al reported that depletion of other apparent Vpr targets, (SMN1, CDCA2 and ZNF267) caused a degree of G2/M arrest. While we cannot exclude the possibility that additional target proteins contribute to the G2/M arrest properties of Vpr, the data presented herein suggests that CCDC137 represents the dominant mediator of the G2/M arrest and HIV-1 gene expression effects of HIV-1 Vpr. Further work, including a detailed analysis of Vpr mutants with distinct target protein specificities should help delineate the relative contributions of various target proteins to Vpr-associated phenotypes. Of note, other viral genes, in particular Vif, are able to affect the cellular environment, including cell cycle perturbation. To exclude the influence of other viral genes, and to facilitate shRNA experiments in primary cells, we employed a minimal version of HIV-1 in most experiments to study the effects of Vpr on cell cycle and gene expression in the target cells. However, we note that the effects of Vpr on gene expression in macrophages were recapitulated with a near full length viral construct, and are consistent with data obtained using a different near full length viral reporter construct reported by Connor et al., 1995. That Vpr associates with a cPERP protein is consistent with prior findings that Vpr binds to chromatin and, with VprBP, forms chromatin-associated nuclear foci, a property that is associated with G2/M cell cycle arrest (Lai et al., 2005;Belzile et al., 2010). CCDC137 is a poorly characterized protein -little is known about its function. CCDC137 has been reported to sequester retinoic acid receptor (RAR) to the nucleolus and thus is dubbed RaRF (Retinoic acid Resistance Factor) (Youn et al., 2018) but whether this property is relevant to HIV-1 gene expression is unknown. Further work will be required to discern the mechanistic details of how CCDC137 affects G2/M transition and inhibits HIV-1 gene expression. Nevertheless, the findings reported herein reveal an important aspect of how HIV-1 manipulates host cells to facilitate its replication.   Representative images of primary macrophages infected with V1/shLuc or V1/shCCDC137II and subjected to fluorescent in situ hybridization (FISH) based detection of HIV-1 RNA using probes directed at the GFP sequence. The FISH signal is displayed in red and GFP protein signal is displayed in green. The upper (V1/shLuc) and lowest two (V1/shCCDC137II) rows are displayed with the same gain and brightness/contrast settings. The second (V1/shLuc) row is a duplicate of the upper row, displayed with enhanced brightness to enable visualization of the FISH and GFP signals. Representative of two experiments, each with three donors. (E) GFP RNA levels (total cell fluorescence) determined by FISH analysis of macrophages infected with V1/shLuc or V1/shCCDC137. Each symbol represents a single cell from a representative donor from one of two experiments, each with three donors. P-value is calculated using a Welch's t-test. (F) qRT-PCR measurement of HIV-1 RNA (GFP) levels in three macrophage donors after infection with V1/shLuc or V1/shCCDC137II. Each symbol Figure 9 continued on next page Continued on next page Figure 9 continued represents a technical replicate. Representative of two experiments, each with three donors. P-value is calculated using a ratio paired t-test for the three displayed donors. (G) qRT-PCR measurement of HIV-1 RNA (Gag) levels in three macrophage donors after infection with V1/shLuc or V1/ shCCDC137II. Each symbol represents a technical replicate. Representative of two experiments, each with three donors. P-value is calculated using a ratio paired t-test for the three displayed donors. The online version of this article includes the following video and figure supplement(s) for figure 9:

Plasmid construction
An HA-epitope was fused in-frame at the N-terminus of HIV-1 NL4-3 Vpr and subcloned into pCR3.1 for transient cotransfection experiments. To express Vpr driven by an HIV-1 long terminal repeat (LTR) in the context of a viral genome, a proviral plasmid was generated harboring a minimal viral genome (V1) (Zennou and Bieniasz, 2006), engineered from HIV-1 NL4/3 (R7/3) harboring large deletions or inactivating mutations in (Gag, Pol, Vif, Vpu and Env) and in which Nef is replaced with GFP. Sequences encoding an HA epitope were fused in frame at N-terminus of Vpr to generate V1/HA-Vpr while V1/d-Vpr was constructed by deletion of the nucleotide sequences between the cPPT/ FLAP and the SalI site within Vpr. In some constructs, the WT HIV-1 NL4-3 Vpr was replaced with Vprencoding sequences from Q65R NL4-3 Vpr, transmitted HIV-1 founder strains, HIV-2, SIV AGM Sab, or SIVmac. To construct the V1/HA-Vpr or V1/d-Vpr expressing mCherry (V1/mCherry), an open reading frame encoding mCherry was amplified, digested with NotI/XhoI, and inserted into V1 to replace GFP. To construct the V1/shCCDC137II carrying an shRNA targeting CCDC137, the DNA sequence containing the U6 promoter and shRNA targeting CCDC137 (I: CAGATGCTGCGGATGCTTCT; II: GGTGAAACATGATGACAACA) was PCR amplified and, after digestion with KpnI, subcloned into V1 carrying inactivating mutations at the 5' end of Vpr (ATGGAACAA/GTGGAATAA), upstream 5' of the RRE (Fig. S6). The control V1/shLuc vector contained the DNA sequence carrying U6 promoter-shRNA targeting luciferase (CGCTGAGTACTTCGAAATGTC) at the same position. V1 derivatives expressing BirA (R118G) and BirA (R118G)-Vpr were constructed by insertion of nucleotide sequences encoding BirA (R118G) or BirA (R118G)-Vpr fusion proteins into the GFP (Nef) position in V1 vector.
Plasmids expressing the various human proteins (CREB1, CREB3L1, hnRNP D, hnRNP F, POC1A, PPM1G, CCDC137, PES1, WDR18, WDR74, MAK16, NOC2L, RRS1, NIP7, hnRNPA1, hnRNP H, hnRNP K, hnRNP R, hnRNP U, hnRNP C, hnRNP A2B1, and DHX9) with a V5 epitope fused to the C-terminus were from a pCSGW-based human ORF lentiviral library. Plasmid pCR3.1 was used to express Bop1, NCL, MKI67IP, or WDR12 with an Flag epitope fused to their N-termini or to express hnRNP A2B1 or DHX9 with an HA epitope fused to their N-termini. Subsequently, CCDC137 was subcloned into pLNCX2 expression vector with an HA epitope fused to its C-terminus. CCDC137 alanine scanning mutants were generated by overlap-extension PCR amplification, using the wildtype CCDC137-HA expression plasmid as the template and inserted into the pLNCX2 expression vector. All aforementioned plasmids were constructed using PCR, Accession numbers for cDNA sequences and oligonucleotides listed in supplementary file 2.
GST-Vpr expression plasmids were based on pCAGGS and were constructed by PCR amplification of the Vpr coding region from HIV-1 NL4-3 which was then inserted in-frame at the 3' end of GST encoding sequences.
To construct a tetracycline-inducible CCDC137 expression vector, wild-type and mutant CCDC137 were amplified, using the corresponding pCR3.1 expression plasmids as templates, and inserted into LKO-derived lentiviral expression vector pLKOD-puro (Busnadiego et al., 2014) which also included a puromycin resistance cassette. All cloned coding sequences were verified by DNA sequencing, oligonucleotide sequences used in construction are listed in supplementary file 2.

Cell lines
Human embryonic kidney HEK-293T (ATCC CRL-3216) were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma F8067) and gentamycin (Gibco). Human bone osteosarcoma epithelial cells (U2OS, ATCC HTB-96) were grown in McCoy's 5a Medium Modified (ATCC 30-2007)/ 10% FCS/gentamycin. MT4 cells (RRID:CVCL_2632) were maintained in RPMI supplemented with 10% fetal bovine serum (FCS) and gentamycin. All cell lines used in this study were monitored by SYBR Green real-time PCR RT assay periodically to ensure the absence of retroviral contamination and stained with DAPI to ensure absence of mycoplasma. Cell line identification was documented by the suppliers. To construct cell-cycle reporter cell line, U2OS cells were transduced with a retroviral vector (pLHCX) encoding Clover (a rapidly-maturing green/yellow fluorescent protein) or mKusabira-Orange2 (mKO2, an orange fluorescent protein) fused to the N-terminus of Geminin 1-110 aa. Single cell clones were isolated after hygromycin selection.

Primary cells
Human lymphocytes were prepared from Leukopaks from NY Blood Center by spinning on top of lymphocyte separation medium (Corning). Macrophages were then isolated by plastic adherence and differentiated using GM-CSF (Thermo Fisher). CD4+ T cells were isolated using an EasySep kit (StemCell) and maintained in RPMI/10%FCS supplemented with IL2.
To test whether Vpr could induce depletion of proteins, 293 T cells in 24-well plates were transfected with 200 ng of pCR3.1-based plasmids expressing Flag-tagged proteins or HA-tagged proteins, or pCSGW-based plasmids expressing V5-tagged proteins (from human ORFs lentiviral library), along with increasing amounts (0 ng, 25 ng, or 50 ng) of a pCR3.1/HA-Vpr expression plasmid. The total amount of DNA was held constant by supplementing the transfection with empty expression vector. Cells were harvested at 28 hr post transfection and subjected to Western blot analysis.
To assess the potency with which HIV-1 Vpr induced CCDC137 depletion, 293 T cells in 24-well plates were transfected with varying amounts (0 ng, 100 ng, 200 ng, or 400 ng/well) of a pCR3.1/ CCDC137-HA expression plasmid and increasing amounts (0 ng, 25 ng, 50 ng, 100 ng, or 200 ng/ well) of a pCR3.1/HA-Vpr expression plasmid. The total amount of DNA was held constant by supplementing the transfection with empty expression vector.

Generation of HIV-1 and lentiviral vector stocks
To generate V1-derived viral stocks, 293 T cells were transfected with 5 mg of pV1-derived proviral plasmids encoding no Vpr or the various HA-tagged Vpr proteins, 5 mg of an HIV-1 Gag-Pol expression plasmid (pCRV1/GagPol) and 1 mg of VSV-G expression plasmid into 293 T cells in 10 cm dishes. Virus-containing supernatant was collected and filtered (0.2 mm) 2 days later. The lentiviral vectors that transduced CCDC137 cDNAs or shRNAs were similarly generated, except that pLKOD-puro or pLKO.1-TRC-derived plasmids were used in place of pV1-derived plasmids.

Immunoprecipitation
HEK-293T cells were transiently transfected with plasmids expressing HA-Vpr and V5-tagged human protein factors, and treated with 10 mM MG132 for 4 hr before harvest and lysis with ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1% digitonin [Sigma], supplemented with 1X complete protease inhibitor [Roche]). After lysis on ice for 10 min, followed by centrifugation at 10,000 rpm for 10 min at 4˚C, clarified lysates were mixed with 1 mg anti-HA monoclonal antibody and rotated with 30 ml pre-equilibrated Protein G Sepharose 4 Fast Flow resin (GE healthcare) for 3 hr at 4˚C. The resin was then washed three times with wash buffer (50 mM Tris, pH 7.4, 150 mM NaCl) and the bound proteins were eluted with SDS-PAGE sample buffer and analyzed by Western blotting.

Glutathione-S-Transferase (GST) fusion protein interaction assay
Human 293 T cells in 6-well plates were co-transfected with 100 ng of GST or 1 mg of GST-Vpr expression plasmids and 500 ng of HA-tagged CCDC137 expression plasmids. The total amount of DNA was held constant by supplementing the transfection with empty expression vector. Two days later, cells were treated with MG 132 for 4 hr and then lysed in Lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, and 1x complete protease inhibitor [Roche]). Cleared lysates were then incubated with glutathione sepharose (GE healthcare) for 4 hr at 4˚C and, after wash with buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100), bound proteins were eluted in SDS-PAGE sample buffer and subjected to Western blot analysis.

BirA-fusion protein interaction assay
Human 293 T cells in 6-well plates were transfected with a V5-tagged CCDC137 expression plasmid and BirA (R118G) or BirA (R118G)-Vpr expression plasmid. Cells were treated with 50 mM biotin at 24 hr after transfection, and 10 mM MG132 at 40 hr post transfection, and harvested at 44 hr post transfection. Cells were then lysed and cleared lysates were incubated with Dynabeads (MyOne Steptavadin C1) above. After thorough wash, biotinylated proteins were eluted from the beads with SDS-PAGE sample buffer supplemented with 200 mM biotin and subjected to Western blot analysis.

Western blot analysis
Cell lysates and immunoprecipitates were separated on NuPage Novex 4-12% Bis-Tris Mini Gels (Invitrogen), and NuPAGE MES SDS running buffer (Invitrogen, NP0002) was used when Vpr was detected. Proteins were blotted onto nitrocellulose membranes. Thereafter, the blots were probed with primary antibodies and followed by secondary antibodies conjugated to IRDye 800CW or IRDye 680. Fluorescent signals were detected and quantitated using an Odyssey scanner (LI-COR Biosciences).

Cell cycle analysis
To determine effects of Vpr on cell cycle, V1 based viral stocks were used to inoculate 2.5 Â 10 5 U2OS cells in 6-well plates at an MOI of 0.5 to 1. At 48 hr post-infection, cells were trypsinized, fixed with paraformaldehyde (PFA) in phosphate-buffered saline (PBS), washed with PBS, and fixed again in 70% ethanol. After an additional wash with PBS, the cells were resuspended in FxCyclePI/RNase Staining Solution (Invitrogen) and incubated at 30˚C for 30 min. Flow cytometric analysis was performed using Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific).
Alternatively, cells were transduced with pLKO.1-TRC-derived vectors encoding shRNA targeting CCDC137. After 48 hr, cells were selected in 1 mg ml À1 puromycin or 5 mg ml À1 blasticidin prior to propidium iodide staining, 40 hr later as described above.
For the experiment in Figure 6, U2OS cells expressing doxycycline-inducible CCDC137 were generated by transduction with a LKO-derived lentiviral vector (Busnadiego et al., 2014) followed by selection in 1 mg ml À1 puromycin. Cells were plated at the density of 2.5 Â 10 5 in 6-well plates in the presence of doxycycline and the next day were infected with V1/HA-Vpr at an MOI of 2 or 0.5. At 12 hr post-infection, doxycycline was replenished and at 48 hr post-infection, cells were harvested for Western blot analysis (for cells infected at high MOI) or cell cycle analysis (for cells infected at low MOI).
For detection of g-H2AX foci, U2OS cells were transduced with lentiviruses encoding CCDC137targeting shRNAs and, after selection with puromycin, cells were seeded on 3.5 cm, glass-bottomed dishes coated with poly-L-Lysine (MatTek). Nuclear foci were visualized by immunostaining with rabbit anti-g-H2AX (abcam, ab11174) followed by a goat anti-rabbit IgG Alexa Fluor-594 conjugate (Invitrogen). A Z-series of images were acquired using an DeltaVision OMX SR imaging system (GE Healthcare).

Live cell microscopy
To monitor cell cycle and HIV-1 (V1) gene expression infection in living cells, U2OS cells expressing mClover-hGeminin (1-110 aa) or primary macrophages were transferred into glass-bottom dishes and time-lapse microscopy was performed using a VivaView FL incubator microscope (Olympus). In some experiments, cells were transduced with lentiviruses containing shRNA targeting CCDC137, 36 hr prior to imaging. In some experiments, cells were infected with V1/d-Vpr or V1/HA-Vpr expressing mCherry or GFP 12 to 24 hr prior to imaging. Images were captured every 30 min using GFP, mRFP and DIC filter sets for up to 72 hr. Preparation of movies was done using MetaMorph software (Molecular Devices) as previously described (Holmes et al., 2015). Images had a depth of 12 bits, that is, an intensity range of 0-4095.

Replicates and statistics
All data is plotted raw, that is individual values for each individual quantitative determination is plotted. The exception to this is CCDC137/Vpr western blot data in Figure 1B, in which the mean of two independent experiments is plotted, with error bars representing the range of the duplicate raw values. Statistical comparisons between groups in Figures 6C, 8H and 9E,F,G. were done using Graphpad Prism software, and p-values were calculated using a Welch's t-test or a ratio t-test.