The ERVK3-1 microprotein interacts with the HUSH complex

Abstract

Human endogenous retroviruses (hERVs) are noninfectious molecular remnants of ancient exogenous retroviruses that now make up 8% of the human genome. The ubiquitously expressed human ERVK3-1 locus was recently annotated as encoding a 109-amino acid endogenous retroviral Rec microprotein. However, because this locus was thought to be non-coding until recently, it is currently unknown whether the ERVK3-1 microprotein has a function in human cells. We demonstrate that the ERVK3-1 microprotein interacts with PPHLN1, a component of the HUSH complex. The HUSH complex promotes transcriptional repression of intron-less genes, which include parasitic genomic elements such as retrotransposons and endogenous retroviruses. We show that the ERVK3-1 microprotein is essential for transcriptional repression of previously identified HUSH target genes. We thus suggest that the ERVK3-1 Rec microprotein contributes to sensing or regulation of target gene expression by the HUSH complex.

Introduction

Human endogenous retroviruses (hERVs) derive from exogenous retroviruses that integrated into the primate germline genome. Inactivation of hERVs is essential to protect the host genome from destabilization that would otherwise result from their active retrotransposition, as well as to circumvent inflammatory signaling downstream of activation of cytoplasmic sensors of viral RNA that results from hERV transcription^1-3. Most hERVs have therefore accumulated mutations that disrupt their protein-coding sequences. As an additional protective mechanism, transcription of hERV and other retroelements is epigenetically repressed, for example by methylation of histone H3 at residue K9 (H3K9me3) by the histone methyltransferase SETDB1 in concert with the HUSH (Human Silencing Hub) complex^4,5. Most hERVs are thus non-coding, though they can exert regulatory effects at the DNA level; for example, some isolated hERV long terminal repeats (LTRs) regulate expression of human genes and hERVs have been identified as STAT1 and IRF1 transcription factor binding sites^6,7. However, despite pressure to mutate and silence hERVs, some hERV protein coding sequences not only remain intact, but have been domesticated to carry out new functions in human cells⁸. A notable example is the HERV-W envelope protein Syncytin-1, which has been repurposed for cell fusion during placenta formation⁹.

In addition to gag, pol, and env coding sequences, some hERVs also encode ancient functional homologs of the human immunodeficiency virus (HIV) Rev protein¹⁰. hERV-encoded Rev homologs have been termed c-orf (central ORF^11,12), K-Rev¹⁰, and, more commonly (and herein), Rec¹³. hERV Rec is a ~100-amino acid protein derived from alternative splicing of the env coding sequence, comprising the first 87 amino acids of Env and a short, hydrophobic C-terminal sequence translated from an alternative reading frame overlapping a downstream region of env^10,14. In the context of the normal retroviral life cycle, Rev/Rec proteins bind to un-spliced and partially spliced retroviral RNA synthesized in the host cell nucleus and recruit Crm1 to export it to the cytoplasm, where new viral particles are assembled¹⁵. hERV Rec proteins bind to a specific structural motif, the 430-nucleotide Rec recognition element (RcRE) within the 3′ LTR to carry out their RNA nucleocytoplasmic transport function, though many hERVs no longer contain functional RcRE motifs^10,11,13.

Materials and Methods

Cell culture, lentivirus production and stable cell line generation

HEK 293T cells were purchased from ATCC, and early-passage stocks were maintained to ensure cell line provenance and sterility. HEK 293T cells were maintained in DMEM (Corning) supplemented with 10% (vol/vol) FBS (Sigma-Aldrich) and 100 U/mL penicillin-streptomycin (VWR) in a 5% CO2 atmosphere at 37°C. Normal human melanocytes (NBMEL) were grown from newborn foreskins in a medium supplemented with bFGF, heparin, IBMX, and dbcAMP exactly as previously described¹⁶.

Cell culture, lentivirus production and stable cell line generation were performed exactly as previously described¹⁷. Briefly, to produce lentiviruses, HEK 293T cells were co-transfected using polyethyleneimine (Polysciences, 23966) with expression construct in pLJM1 along with pMD2.G and psPAX2, and growth medium was replaced after 7–8 h. 48 h after transfection, medium containing viruses was collected and filtered through a 0.45-μm filter, and infection was performed by mixing with two volumes of fresh medium containing suspended HEK 293T cells. Twenty-four hours after infection, the growth medium was replaced. Forty-eight hours after infection, stable cells were selected with 4 μg/mL puromycin for 2 days. Early stocks of stable cell lines were established after selection. Stable cell lines were released from puromycin for 2 days before use in experiments.

Antibodies

Primary antibodies for Western blotting include anti-FLAG (1:1000, Sigma, F1804), anti-β-actin (1:3000, Invitrogen, MA5-15739), anti-Histone-H3 (1:2000, Cell Signaling, 4499), anti-PPHLN1 (1:1000, Abcam, ab69569), anti-ERVK3-1 (1:1000). Secondary antibodies for western blotting were goat anti-rabbit IgG horseradish peroxidase conjugate (1:4,000, Rockland, 611-1302) and goat anti-mouse IgG horseradish peroxidase conjugate (1:4,000, Rockland, 610-1319). Primary antibodies/antisera for immunostaining were mouse anti-FLAG (1:1000, Sigma, F1804) and in-house generated rabbit anti-ERVK3-1 (1:2000, see below). Secondary antibodies for immunostaining were goat anti-mouse IgG Alexa Fluor 647 (1:500, Invitrogen, A21235), and goat anti-rabbit IgG Alexa Fluor 647 (1:1000, Invitrogen, A21244).

Antibody generation

All animal procedures received approval from the Institutional Animal Care and Use Committee of the Salk Institute and adhered to the PHS Policy on Humane Care and Use of Laboratory Animals (PHS Policy, 2015), the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training, the NRC Guide for Care and Use of Laboratory Animals (8th edition), and the USDA Animal Welfare Act and Regulations. Animals were housed in an AAALAC accredited facility in a climate-controlled environment under 12 hr light/12 hr dark cycles. Rabbits were provided ad libitum feed (5326 Lab Diet, high fiber), microfiltered water, and weekly fruits and vegetables and alfafa hay for enrichment. Animals were monitored daily by the veterinary staff for good health. Three specific-pathogen free female New Zealand white rabbits aged 12 to 14 weeks, weighing 3.0 to 3.2 kg at the study's commencement, and sourced from Western Oregon Rabbit Co. (WORC, Philomath, OR, USA), were used for human ERVK3-1 antisera production.

A synthetic peptide fragment encoding human ERVK3-1 (55-86), with the addition of a cysteine and spacer at the amino terminus, CG-TSNPITWGQIKKTTQEAEKLLERQGQAKTPDS, was coupled to keyhole limpet hemocyanin (KLH) via maleimide, following manufacturer’s instructions (ThermoFisher, Waltham MA). The peptide was synthesized, C18 HPLC purified to 90%, and amino acid sequence verified by MS by RS Synthesis (Louisville, KY). The immunogen was prepared by emulsification of Freund’s complete adjuvant-modified Mycobacterium butyricum (EMD Millipore) with an equal volume of phosphate buffered saline containing 1.0 mg conjugate/ml for the first two injections. For booster injections, incomplete Freund’s adjuvant was mixed with an equal volume of PBS containing 0.5 mg conjugate/ml. For each immunization, an animal received a total of 1 ml of emulsion in 20 intradermal sites in the lumbar region, 0.5 mg total protein conjugate for the first two injections and 0.25 mg total protein conjugate for all subsequent booster injections. Three individual rabbits were injected every three weeks and were bled one week following booster injections, <10% total blood volume. Rabbits were administered 1-2 mg/kg Acepromazine IM prior to injections of antigen or blood withdrawal. At the termination of study, rabbits were exsanguinated under anesthesia (ketamine 50 mg/kg and aceprozamine 1 mg/kg, IM) and euthanized with an overdose of pentobarbital sodium and phenytoin sodium (1 ml/4.5 kg of body weight IC to effect). After blood was collected the death of animals was confirmed. All animal procedures were conducted by experienced veterinary technicians, under the supervision of Salk Institute veterinarians.

Each bleed from each animal was tested at multiple doses for the ability to recognize the synthetic peptide antigen. Bleeds exhibiting the highest titers underwent further analysis through Western immunoblot to confirm their ability to recognize the recombinantly expressed ERVK3-1 microprotein. The HERVK3-1 antiserum derived from the rabbit displaying optimal characteristics in terms of titer against the synthetic peptide antigen and the ability to recognize the endogenous protein was utilized in all experiments.

Cloning and genetic constructs

The ERVK3-1 coding sequence was subcloned with an N-terminal FLAG epitope tag into pLJM1. The FLAG-ERVK3-1 construct was used to generate Figure 2. A separate construct encoding the coding sequence of ERVK3-1 with a V5 tag at the 5’ end of the coding sequence was subcloned into pLJM1. This construct was used to generate stable rescue cell lines. For proximity labeling, the ERVK3-1 coding sequence was subcloned downstream of TurboID-V5 into pLJM1. All plasmids are available upon request.

Figure 2.

ERVK3-1 interacts with periphilin-1 (PPHLN1). (A) Volcano plot of quantitative proteomics (N = 3 biologically independent experiments) of streptavidin pull-down from nuclear lysates of biotin-labeled HEK 293T cells stably expressing TurboID-ERVK3-1 versus wild type HEK 293T cells. Significance (unpaired t-test, p < 0.05) was calculated using Perseus. Horizontal dotted line signifies cutoff of 1.3, and vertical dotted lines signifies a cutoff of 20. (B) Volcano plot of quantitative proteomics (N = 3 biologically independent experiments) for co-immunoprecipitation of FLAG-ERVK3-1 expressed in HEK 293T cells versus wild type HEK 293T cells. Significance (unpaired t-test, p < 0.05) was calculated using Perseus. Horizontal dotted line signifies cutoff of 1.3, and vertical dotted lines signifies a cutoff of 100. (C) Validation of ERVK3-1 interaction with PPHLN1 by anti-FLAG co-immunoprecipitation (co-IP) from HEK 293T cells stably expressing ERVK3-1-FLAG cells and Western blotting. Cell lysates (2%) before co-IP (input) were used as loading controls. (D) The relative abundance of ERVK3-1 in cytoplasmic vs. nuclear fractions of HEK 293T cells was assessed by Western blotting with anti-ERVK3-1 antiserum. Absence of cytoplasmic proteins from the nuclear fraction was confirmed with anti-tubulin Western blotting. (E) Immunostaining of HEK 293T cells with an antiserum against ERVK3-1 (magenta) and DAPI (cyan). Scale bars, 10 μm.

Human subjects

Peripheral blood mononuclear cells (PBMCs) were isolated from blood drawn from a healthy donor as previously described¹⁸. Informed consent was obtained from the subject. These studies were approved by the institutional review committee of Yale University (JC).

Microprotein enrichment and digestion

To detect microproteins in HEK 293T, NBMEL, and PBMCs, cell pellets were lysed and small proteome fractions were excised from Tris-Tricine SDS-PAGE gels followed by in-gel trypsin digest exactly as previously described^19,20.

Biotin labeling with TurboID in mammalian cells

For labeling of HEK 293T V5-TurboID-ERVK3-1 and wild-type HEK 293T (negative control) cell lines (grown in 15 cm dishes), a final concentration of 500 μM biotin for 30 min at 37 °C was used following a reported protocol²¹. Labeling was stopped after the desired time by transferring the cells to ice and washing five times with cold PBS buffer. Cells were detached from the flask by gently pipetting of PBS directly onto the cells, then pellets were collected by centrifuging the resulting cell suspension at 1,600 r.p.m. for 5 min. Cells were suspended in 1 mL nuclear isolation buffer (10 mM Hepes pH 7.4, 100 mM KCl, 5 mM MgCl₂ with 0.5% NP40 and Roche Complete protease inhibitor cocktail tablets (Roche, Cat. No.11873580001), and incubated on ice for 10 min, followed by centrifugation 3 min at 4°C by 3,000 rpm. The nuclear pellets were suspended in 1 mL RIPA lysis buffer by gentle pipetting and sonication at 4 °C. Lysates were clarified by centrifugation at 15,000 r.p.m. for 30 min at 4 °C. To enrich biotinylated proteins, 300 μL Dynabeads^™ M-280 Streptavidin (Thermo Fisher, Cat. #11205D) were washed twice with RIPA buffer, incubated with cell lysates containing ~10 mg protein for each sample and rotated at 4 °C overnight. The beads were subsequently washed twice with 1 mL of RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na₂CO₃, once with 1 mL of 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice with 1 mL RIPA lysis buffer. Bound proteins were eluted by boiling in 2×SDS loading buffer containing 20 mM DTT and 2 mM biotin for 15 min.

The eluted proteins were subjected to SDS-PAGE separation prior to LC-MS/MS analysis as previously described²⁰. Briefly, gel slices containing entire lanes were digested with trypsin at 37°C for 14–16 h. The resulting peptide mixtures were extracted from the gel, dried, subjected to ethyl acetate extraction to remove residual detergent, de-salted with peptide cleanup C18 spin column (Agilent Technologies, 5188-2750), then resuspended in 35 μL 0.1% formic acid (FA), followed by centrifugation at 21,130 g, 4°C, 30 min. A 5 μL aliquot of each sample was injected onto a pre-packed column attached to a nanoAcquity UPLC (Waters) in-line with a Thermo Scientific Q Exactive Plus Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific).

For proximity labeling proteomics searches and quantitative analysis, files were analyzed using MaxQuant, cysteine carbidomethylation was set as a fixed modification, oxidation of methionine and N-terminal acetylation were set as variable modifications, and human UniProt was used as the database for searching. For all analysis, a mass deviation of 20 p.p.m. was set for MS1 peaks, and 0.02 Da was set as maximum allowed MS/MS peaks with a maximum of two missed cleavages. Maximum false discovery rates (FDR) were set to 1% both on peptide and protein levels. Minimum required peptide length was five amino acids. Protein quantitation was accomplished by calculating the LFQ intensity ratio of TurboID-ERVK3-1 pulleddown to corresponding negative control samples using MaxQuant (version 1.6.8.0) with standard parameters. p values (two-sample t test) were calculated using Perseus (version 1.5.8.5) with standard parameters.

Microprotein mass spectrometry

Proteomic analysis of microproteins was performed exactly as previously described²⁰. Identification of microproteins was performed using the Mascot search engine coupled with specialized databases generated from three-frame translation of RNA-seq data from similar samples (e.g., HEK 293T, PBMCs, melanoma cells for NBMLC) exactly as previously described²⁰; search databases are available upon request.

Co-immunoprecipitation (co-IP), subcellular fractionation and Western blotting

HEK 293T cells stably expressing FLAG-tagged ERVK3-1 coding sequence, or wild-type HEK 293T controls, were grown in 15-cm dishes, harvested and lysed using Tris-buffered saline (TBS) with 1% Triton X-100 and Roche Complete protease inhibitor cocktail tablets. 400 μL lysis buffer was used per pellet. Cells were lysed on ice for 20 min followed by centrifugation at 14000 rpm, 4°C, 15 min. Lysate samples were saved for analysis of loading. A 50 μL aliquot of anti-FLAG agarose beads (clone M2, Sigma) was washed with 1 mL TBS-T, collected by centrifugation for 1 min at 3000 rpm, then suspended in the cell lysate supernatant. Bead suspensions were rotated at 4°C for 1 hour, then washed 3 times with TBS-T. Elution was in 40 μL of 3× FLAG peptide (Sigma), in TBS-T at 4°C for 1 hour, after which beads were removed by centrifugation.

1% of cell lysate and 50% of the immunoprecipitates were mixed with protein loading buffer, boiled, and separated on 4–20% Tris/glycine SDS-PAGE gels (BioRad). Proteins were transferred to nitrocellulose membranes for 2 h at 400 mA. Immunoblots were blocked with blocking buffer (3% BSA in 1X TBST), then probed with primary antibodies at a 1:1000 dilution in the same buffer for ~2 hours at 4°C. The membrane was washed three times with TBS-T. For chemiluminescence imaging, secondary antibodies were applied at a dilution of 1:10,000 in 3% BSA in TBS-T, then washed 3× with TBS-T prior to development with Clarity ECL Western Blotting Substrate (Bio-Rad) and imaged using a UVP ChemiDoc-It Imaging System with Visionworks Software.

For subcellular fractionation prior to Western blotting, HEK 293T cells were pelleted, flash frozen, and processed with the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific catalog number 78833) per the manufacturer’s instructions.

Immunofluorescence

For imaging, HEK 293T cells were plated on glass coverslips pre-treated with fibronectin (Millipore, 341635) following manufactures’ guidelines. Cells were cultured overnight to 70–80% confluency in 12-well plate, fixed in 10% formalin for 15 min at room temperature (RT), washed with PBS once, then permeabilized with PBS containing 0.2% (v/v) Triton X-100 (PBST). After rinsing 3 times with PBST for 5 minutes each, the cells were blocked with 1% BSA in PBST at room temperature for 1 hour. Afterwards, the cells were incubated with primary antibodies overnight at 4°C. After rinsing 3 times with PBST, the cells were incubated with secondary antibodies and DAPI for 1 hour at RT, washed with PBST and mounted with Mowiol (Sigma, 81381) before viewing, or were imaged directly after staining. Confocal imaging was performed on a Leica SP8 LS confocal microscope with a 63× oil immersion objective. The images were processed with ImageJ (Version 1.5m).

Generation of ERVK3-1 knock-out (KO) cell lines

A clonal ERVK3-1 KO HEK 293T cell line was generated using CRISPR-Cas9. Guide RNAs (gRNAs) were designed with the guide design tools from https://portals.broadinstitute.org/gppx/crispick/public to target the ERVK3-1 genomic region (gRNA1: 5’-TGTACCCGCTCAATAATGCC-3’, gRNA3: 5’-TTGAATACCATGGATCCCTC-3’), or a non-targeting gRNA (5’-GTACGTCGGTATAACTCCTC-3’). Double-stranded DNA oligonucleotides corresponding to the gRNAs were inserted into pSpCas9(BB)-2A-GFP vector (Addgene, as a gift from F. Zhang, MIT).

For generation of clonal KO cells, an equal mixture of the two gRNA plasmids, or non-targeting gRNA plasmid, was transfected into HEK 293T cells using polyethyleneimine, and single GFP-positive cells were sorted with flow cytometry. The ERVK3-1 deletion was confirmed by genomic DNA PCR, followed by Sanger sequencing.

qRT-PCR

Total RNA was extracted from wild-type HEK 293T, HEK 293T in which Cas9 and a non-target guide RNA were expressed, HEK 293T ERVK3-1 KO, and HEK 293T ERVK3-1-V5 rescue cells with TRIzol (Cat. No. 15596026; Life Technologies) per the manufacturer’s instructions. After RNA treatment with DNase I (NEB Cat. No. M0303S), cDNA was synthesized using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad, Cat. 172-5035) following the manufacturer’s instructions. Reactions for qPCR were set up on ice according to the manufacturer’s instructions using the iTaq Universal SYBR Green Supermix (Bio-Rad, Cat. 172-5121). Amplification of beta-actin RNA was used as an internal control, and relative expression between samples was calculated with the comparative CT (2−ΔΔCt) method as previously reported²².

RNA-Seq

Total RNA was extracted using RNEasy Kit (Qiagen, Cat. No. 74104) and treated with Turbo DNase I (NEB Cat. No. M0303S) according to the manufacturer’s protocol. Stranded RNA-seq libraries were prepared and sequenced on the Illumina NovaSeq 6000 platform (150 bp paired-end) at the Yale Center for Genome Analysis (YCGA), generating over 40 million reads per sample across three biological replicates. Raw reads were assessed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adapter sequences were trimmed using cutadapt (v4.6). Filtered reads were aligned to the human reference genome (GRCh38) using STAR (v2.7.10a) in two-pass mode, with modified parameters (--outFilterMultimapNmax 100, --winAnchorMultimapNmax 200) to enhance multi-mapping performance over repetitive regions. To facilitate normalization, 5% Drosophila melanogaster total RNA was added to each sample prior to library construction as an external spike-in control. Reads were simultaneously aligned to the Drosophila reference genome (BDGP6.54), and the number of spike-in–mapped reads was used to compute normalization factors for cross-sample scaling. Gene expression quantification was performed using featureCounts (v2.0.8)¹¹ with Ensembl GRCh38.109 annotations. Differential expression analysis was conducted using DESeq2 (v1.36.0)¹², incorporating spike-in–derived scaling factors. Transposable element (TE) expression was quantified using TEtranscripts (v2.2.3), leveraging RepeatMasker annotations from the UCSC Genome Browser. Both uniquely and multi-mapped reads were retained. Locus-specific quantification of human endogenous retroviruses (ERVs) was performed using the ERVmap annotation, a manually curated dataset of transcribed ERV loci derived from the literature and optimized for RNA-seq analysis². Instead of employing the original ERVmap pipeline, the annotation was integrated with featureCounts to obtain locus-level read counts, which were then used for differential expression analysis with DESeq2. All downstream analyses were conducted in R (v4.2.3).

ChIP-qPCR

Cas9 control, ERVK3-1 KO and rescue HEK 293T cells were grown to 80–90% confluency in 10-cm dishes. The cells were washed with 2.5 mL PBS once, then incubated with 4 mL 1% (v/v) formaldehyde (Sigma, 252549) in PBS for 10 min at RT for cross-linking, and quenched by adding 0.2 mL 2.5 M glycine in PBS at RT for 5 min. Cells were washed twice with cold PBS, collected, aliquoted into two 1.5-mL tubes per 10-cm dish and flash frozen for later use. Collected cells from one 1.5-mL tube were resuspended in 150 μL buffer 1 (15 mM Tris-HCl pH 7.5, 60 mM KCl, 5 mM MgCl₂, 0.1 mM EGTA, 0.3 M sucrose), then combined with 150 μL buffer 2 (15 mM Tris-HCl pH 7.5, 60 mM KCl, 5 mM MgCl₂, 0.1 mM EGTA, 0.3 M sucrose, 0.5% NP-40 (v/v), 1% NaDOC (w/vol)) and incubated on ice for 15 min, then combined with 300 μL buffer 3 (85 mM Tris-HCl pH 7.5, 3 mM MgCl₂, 2 mM CaCl₂, 0.3 M sucrose), and incubated at 37°C for 5 min. 1.0 μL MNase was added and incubated at 37°C for 15 min, then quenched by adding 6 μL 0.5 M EDTA-KOH pH 7.5, and 6 μL 100x Roche Complete protease inhibitor cocktail tablets (Roche, 11873580001), followed by sonication (30% intensity, 5 s pulse with 25 s rest, 3 cycles, MICROSON XL 2000) on ice and centrifugation at 13,523 g, 4°C, 10 min.

The DNA content in each supernatant was measured and normalized to 0.17 μg/μL with ChIP buffer (50 mM Tris-HCl pH 7.5, 30 mM KCl, 4 mM MgCl₂, 1 mM CaCl₂, 0.05 mM EGTA, 5 mM EDTA, 0.3 M sucrose, 0.125% NP-40 (v/v), 0.25% NaDOC (w/vol)). 500 μL of the digested lysates were immunoprecipitated with anti-H3K9me3 (1:50, CST, 13969S) overnight at 4°C, followed by incubating with 25 μL of Dynabeads Protein A (Invitrogen, 10001D) for 1 h at 4°C. The beads were washed once with 1 mL low salt buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 0.1% SDS (w/vol), 1% Triton X-100 (v/v)), 1 mL high salt buffer (20 mM Tris-HCl pH 8.1, 500 mM NaCl, 2 mM EDTA, 0.1% SDS (w/vol), 1% Triton X-100 (v/v)), 1 mL LiCl buffer (50 mM Tris-HCl pH 8.1, 1 mM EDTA, 0.25 M LiCl, 1% NP40 (v/v), 1% NaDOC (w/vol)), 1 mL ×2 TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).

The DNA and proteins were eluted by adding 200 μL elution buffer (1% SDS, 0.1 M NaHCO₃ in ddH₂O). After rotating at room temperature for 15 min, the supernatant was transferred to new tubes. 20 μL of the digested lysates were diluted by adding 180 μL elution buffer as input. Crosslink reversal was performed by adding 8 μL 5 M NaCl and incubating at 65°C overnight. RNAs were removed by adding 1 μL RNase A (Thermo Fisher, EN0531), and incubated at 37°C for 1 h. Proteins were digested by adding 2 μL proteinase K (NEB, P8107S), 8 μL 1 M Tris-HCl pH 6.5, 4 μL 0.5 M EDTA, and incubated at 55°C for 2 h. The DNAs were extracted by adding 1 mL PB buffer (5 M guanidium HCl, 30% isopropanol (v/v)), and passed through a spin column for DNA (Epoch Life Science, 1910-250) following standard protocols for DNA purification. Quantitative ChIP-PCR was performed on selected genes with Luna Universal qPCR Master Mix (NEB #M3003).

Conservation analysis

ERVK3-1 orthologs from selected primates were manually analyzed, including NP_001383009.1 (human, Homo sapiens), XP_054108643.1 (white-tufted-ear marmoset, Callithrix jacchus), XP_045236469.1 (crab-eating macaque, Macaca fascicularis), XP_055095933.2 (siamang, Symphalangus syndactylus), XP_055226837.1 (western lowland gorilla, Gorilla gorilla gorilla), XP_054959953.2 (pygmy chimpanzee, Pan paniscus), XP_054529594.1 (chimpanzee, Pan troglodytes), XP_054320953.1 (bornean orangutan, Pongo pygmaeus), XP_054395203.1 (sumatran orangutan, Pongo abelii), XP_058281797.1 (silvery gibbon, Hylobates moloch), and XP_050624657.1 (tibetan macaque, Macaca thibetana thibetana). The sequences were aligned with Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo).

Quantification and statistical analysis

All values and error bars represent the mean ± SEM. Significance was evaluated by a two-tailed t test using GraphPad Prism (version 10.4.1). p values (t test) for proteomics were calculated using Perseus (version 1.5.8.5) with standard parameters, and p value ≤ 0.05 was considered significant.

Results and Discussion

Several proviruses from the HERV-K family (namely from the HML-2, HML-6, HML-10 clades) have largely retained their protein coding capacity^12,14,23. Notably, many hERV-Ks encode Rec proteins that differ slightly in amino acid sequence, and several are transcribed²³. Uniquely among HERV-Ks, ERVK3-1 is ubiquitously expressed in differentiated human cells and tissues. Consistent with evidence for its transcription²³, coding capacity²³, and protein-level expression²⁴, ERVK3-1 was recently annotated as encoding a 109-amino acid Rec protein (NCBI Reference Sequence: NP_001383009.1), which is conserved in some primates (Figure 1A) and is predicted to be structured²⁵ (Figure 1B). We refer to ERVK3-1 as a microprotein, defined as a short coding sequence previously missing from the human genome annotation^26,27. We previously detected expression of the ERVK3-1 microprotein (hereafter, ERVK3-1) in leukemia-derived cell lines²⁴, and we hypothesized that it may also be expressed at the protein level in normal cells. Using a previously reported proteomic method^20,28, we detected a unique tryptic peptide supporting ERVK3-1 expression in HEK 293T cells, primary human melanocytes from a healthy donor, and peripheral blood mononuclear cells from a healthy donor (Figure S1).

Figure 1.

ERVK3-1 microprotein conservation and structure. (A) Clustal Omega alignment of primate ERVK3-1 microprotein sequences. (B) Predicted three-dimensional structure of ERVK3-1, obtained from the AlphaFold Protein Structure Database and colored according to confidence score. Dark blue: Very high (pLDDT > 90); Cyan: High (90 > pLDDT > 70); Yellow: Low (70 > pLDDT > 50); Orange: Very low (pLDDT < 50). pLDDT: predicted Local Distance Difference Test.

To further validate endogenous expression of the ERVK3-1 microprotein in human cells, we raised an antiserum against ERVK3-1. We identified a specific immunoreactive band at the expected size in wild-type HEK 293T cells that was correspondingly increased in HEK 293T cells stably overexpressing ERVK3-1, and absent in knockout (KO) HEK 293T cells in which the ERVK3-1 coding sequence was disrupted with CRISPR/Cas9 (Figure S2A-B). These results support protein-level expression of the 109-amino acid ERVK3-1 microprotein in primary and immortalized human cells from varied tissues of origin.

Given its ubiquitous expression, we hypothesized that the ERVK3-1 microprotein may function in human cells. Many microproteins interact with proteins or macromolecular complexes²⁹. In order to determine ERVK3-1 interaction partners, we first generated a HEK 293T cell line stably expressing ERVK3-1 as a fusion to the proximity biotinylation enzyme TurboID³⁰, because proximity labeling has been previously shown to increase the specificity of microprotein interaction partner detection relative to co-immunoprecipitation (co-IP)³¹. We confirmed biotinylation activity of the ERVK3-1-TurboID fusion construct, and differential proteome labeling compared to a nuclear-localized control TurboID construct (TurboID-NLS, Figure S3A). Streptavidin enrichment of ERVK3-1-TurboID-labeled proteins followed by quantitative proteomic analysis in comparison to a negative control revealed that ERVK3-1 associated with two HUSH complex members, the core component periphilin 1 (PPHLN1) and the accessory factor ZNF638 (also called NP220³²) as well as several other chromatin-associated proteins (Figure 2A). To orthogonally identify potential ERVK3-1 interactors, we performed co-IP. FLAG-tagged ERVK3-1 was co-immunopurified from HEK 293T cells, and proteins enriched over parental HEK 293T cell controls were identified with label-free quantitative proteomics (Figure 2B). PPHLN1 was again one of two highly enriched proteins; the other, LACRT, is secreted and likely does not encounter ERVK3-1 in intact cells, demonstrating the non-specificity inherent in co-IP.

Since it was the only ERVK3-1-proximal protein identified by two experimental approaches, we selected PPHLN1 for validation. First, we performed co-IP of stably expressed ERVK3-1-FLAG from HEK 293T cells followed by Western blotting (Figure 2C). PPHLN1 was enriched in the ERVK3-1 co-IP and not in the control, validating their association. Finally, to provide additional evidence to support the interaction of ERVK3-1 with nuclear proteins, we examined the subcellular localization of endogenous ERVK3-1. We observed a subpopulation of endogenous ERVK3-1 in the nucleus as well as the cytoplasm via subcellular fractionation and Western blotting (Figure 2D) and by immunofluorescence (Figure 2E). We concluded that ERVK3-1 associates with PPHLN1, though we cannot exclude the possibility that ERVK3-1 may also interact with additional proteins inside or outside the nucleus.

PPHLN1, along with MPP8 and TASOR^33,34, is a component of the HUSH complex. HUSH recognizes intron-less transcripts derived from young, actively retrotransposing LINE-1 elements, retroviruses, processed pseudogenes and Krüppel-associated box-zinc-finger protein (ZNF) genes to promote H3K9me3 deposition and transcriptional repression of these loci^33,35. We hypothesized that ERVK3-1 could be involved in repression of HUSH target genes due to its interaction with PPHLN1. To test this hypothesis, we first examined whether ERVK3-1 affects PPHLN1 stability, but no change in PPHLN1 levels was observed in ERVK3-1 KO cells (Figure S3B). We next examined whether ERVK3-1 affects gene expression, with a focus on known HUSH target genes. To do this, we performed RNA-seq to measure global gene expression changes in ERVK3-1 KO cells compared to HEK 293T expressing Cas9 with a non-target guide RNA (Figure 3 and S4A). We examined genes upregulated with statistical significance in the KO cells, which could represent both de-repressed genes as well as secondary gene expression changes. Importantly, a number of pseudogenes and zinc finger proteins (ZNFs) were upregulated, which are known classes of HUSH targets, including specific previously reported examples ZNF233 and ZNF229^34,36. Furthermore, a number of genes involved in inflammation and interferon signaling were also upregulated, in line with previous reports that loss of HUSH activity leads to interferon signaling³⁷. While they are not previously reported HUSH targets, we noticed that some of the most highly upregulated genes in our dataset encode MAGE (melanoma antigen gene) proteins. MAGE genes are normally heavily CpG methylated and silenced in somatic tissues, though they can be aberrantly upregulated in cancer³⁸. Finally, GSTF1 was strongly upregulated. GTSF1 normally functions in the piRNA-PIWI pathway to silence transposons in germ cells³⁹, which may be consistent with compensatory upregulation of alternative pathways to silence TEs in the ERVK3-1 KO. We separately processed the RNA-seq data to assess changes in transposable elements (TEs⁴⁰, Figure S4B) and hERVs² (Figure S4C). Few changes in TE expression were observed, in line with selective targeting of a subset of evolutionarily young transposons by HUSH⁴¹, while hERV expression was dysregulated in both directions. Overall, widespread gene expression changes occur in ERVK3-1 KO cells, many of which are consistent with a function for ERVK3-1 in the HUSH complex.

Figure 3.

HUSH (Human Silencing Hub) target genes and genes involved in immune recognition and signaling are upregulated in ERVK3-1 knockout (KO) cells. Volcano plot of RNA-Seq analysis showing transcriptional changes (upregulation by 2-fold or higher) in protein coding genes in ERVK3-1 KO compared to HEK 293T cells expressing Cas9 with a non-target guide RNA. Genes selected for further validation are indicated. Data points not included in this figure (axis break) can be found in Figure S4A. Data represent 3 biologically independent samples. Significance, Student’s t test (p < 0.05).

We next sought to further examine ERVK3-1-dependent regulation of HUSH target gene expression with qRT-PCR in ERVK3-1 KO cells, with comparison to control cells subjected to Cas9 expression in the presence of a non-targeting guide RNA, and to conclusively establish the direct dependence of this regulation on the ERVK3-1 Rec microprotein using rescue cells in which the ERVK3-1 coding sequence was stably reintroduced into the KO cells. A subset of LINE-1s (L1s) are well-validated HUSH complex targets³⁷, so we performed qRT-PCR of L1ORF2 as a reporter to determine if ERVK3-1 affects its expression. We observed ~5-fold upregulation of L1ORF2 in ERVK3-1 KO cells relative to control cells, while ERVK3-1 rescue decreased L1ORF2 to levels comparable to wild-type (Figure 4A, left). In addition, we validated upregulation of previously identified HUSH target genes ZNF229 and ZNF233 (Figure 3) in ERVK3-1 KO, as well as repression of these genes in ERVK3-1 rescue cells (Figure 4A, center and right). Expression changes in MAGE A3/B2 and GTSF1, which we observed in RNA-seq (Figure 2), were also ERVK3-1 microprotein-dependent by qRT-PCR (Figure 4B). Importantly, ERVK3-1 mRNA levels do not change significantly in KO or rescue cells relative to controls, distinguishing protein- vs. RNA-level function of ERVK3-1 (Figure S5A).

Figure 4.

ERVK3-1 is required for repression of selected HUSH complex target genes. (A) The expression of HUSH complex target genes L1ORF2, ZNF229, and ZNF233 were measured by quantitative RT-PCR (qRT-PCR) with reference to beta-actin in HEK 293T cells that expressed Cas9 with a non-target guide RNA (Control), ERVK3-1 knockout (KO) HEK 293T cells, and rescue cells in which ERVK3-1-V5 was stably reintroduced in ERVK3-1 KO cells (Rescue). (B) Selected genes identified as differentially expressed in ERVK3-1 KO by RNA-Seq (Figure 2) were validated via qRT-PCR. (C) H3K9 trimethylation at HUSH target genes was measured with quantitative ChIP-PCR (ChIP-qPCR) in control, KO and rescue cell lines. Individual data points are from 3 (qRT-qPCR) or 4 (ChIP-qPCR) technical replicates and error bars represent mean values ± SEM. Significance (p value) was evaluated with unpaired t test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

To determine whether the observed changes in previously reported HUSH target gene expression in ERVK3-1 KO cells were a result of changes in HUSH activity, we used chromatin immunoprecipitation (ChIP)-qPCR to measure H3K9me3 levels³⁵ at these loci. In all cases, a statistically significant decrease in H3K9me3 levels was observed in ERVK3-1 KO, and partial or complete restoration of wild-type methylation occurred when the ERVK3-1 coding sequence was reintroduced (Figure 4C, Figure S5B). We conclude that the ERVK3-1 Rec microprotein contributes to H3K9me3 methylation and transcriptional repression of a subset of HUSH complex target genes.

Conclusion

In summary, the ERVK3-1 Rec microprotein associates, both physically and functionally, with the HUSH complex, and transcriptional repression of several well-validated HUSH target genes depends at least in part on ERVK3-1. Due to its previous annotation as a non-coding locus²³, the ERVK3-1 microprotein would have been undetectable in previous attempts to identify HUSH complex members³⁴. Our data clearly implicate ERVK3-1 in HUSH complex activity, though its molecular mechanism remains to be established. It is possible that ERVK3-1 contributes to allosteric activation or assembly of the HUSH complex. Alternatively, ERVK3-1 could participate in HUSH target recognition through RNA binding (e.g., of specific RcRE-containing nascent transcripts) – a recently described property of HUSH ascribed to PPHLN1⁴² – since, as described above, this is the native function of Rec proteins in the retroviral life cycle. It is alternatively possible that ERVK3-1 binds to PPHLN1 and exerts its effects independent of the rest of the HUSH complex. Furthermore, given the reported encoding of Rev/Rec homologs in multiple transcribed HERV-K loci^8,14, as well as in exogenous retroviruses, it will be critical to determine whether the ERVK3-1 microprotein is unique in its association with PPHLN1 and contribution to HUSH target gene repression, or whether this is a general property of Rec and Rec-like proteins, which may be encoded in multiple hERV loci. Regardless, our work demonstrates that ERVK3-1 represents a novel example of a hERV Rec protein that has acquired a function in human cells and suggests that many more functional microproteins may remain to be discovered in non-coding genomic regions.

Supplementary Material

Supporting Information. Figures S1-S4 detailing validation of ERVK3-1 knockout (KO) cell lines, validation of ERVK3-1-TurboID proximity biotinylation, and measuring mRNA levels in KO and rescue cells; Table S1, sequences of primers used in this work. (PDF)

ABBREVIATIONS

gRNA

guide RNA

H3K9me3

trimethylation of histone H3 at residue K9

HEK

human embryonic kidney

hERV

human endogenous retrovirus

HUSH

human silencing hub

KO

knockout

LINE

long interspersed nuclear element

MS/MS

tandem mass spectrometry

NBMELC

newborn melanocytes

PBMC

peripheral blood mononuclear cells

PPHLN1

periphilin-1

(q)RT-PCR

reverse transcription and quantitative polymerase chain reaction

SEM

standard error of the mean

UV-vis

UV-visible spectroscopy

DATA AVAILABILITY

Proteomics data reported in this study are available via PRIDE (Proteomics Identifications Database) under project accession PXD064459. The RNA-seq data reported herein are posted under NCBI Gene Expression Omnibus GSE300870. Plasmids utilized in this work are available upon request.