Predicted Cellular Interactors of the Endogenous Retrovirus-K Integrase Enzyme

Abstract

Integrase (IN) enzymes are found in all retroviruses and are crucial in the retroviral integration process. Many studies have revealed how exogenous IN enzymes, such as the human immunodeficiency virus (HIV) IN, contribute to altered cellular function. However, the same consideration has not been given to viral IN originating from symbionts within our own DNA. Endogenous retrovirus-K (ERVK) is pathologically associated with neurological and inflammatory diseases along with several cancers. The ERVK IN interactome is unknown, and the question of how conserved the ERVK IN protein–protein interaction motifs are as compared to other retroviral integrases is addressed in this paper. The ERVK IN protein sequence was analyzed using the Eukaryotic Linear Motif (ELM) database, and the results are compared to ELMs of other betaretroviral INs and similar eukaryotic INs. A list of putative ERVK IN cellular protein interactors was curated from the ELM list and submitted for STRING analysis to generate an ERVK IN interactome. KEGG analysis was used to identify key pathways potentially influenced by ERVK IN. It was determined that the ERVK IN potentially interacts with cellular proteins involved in the DNA damage response (DDR), cell cycle, immunity, inflammation, cell signaling, selective autophagy, and intracellular trafficking. The most prominent pathway identified was viral carcinogenesis, in addition to select cancers, neurological diseases, and diabetic complications. This potentiates the role of ERVK IN in these pathologies via protein–protein interactions facilitating alterations in key disease pathways.

1. Introduction

Viral proteins often usurp and alter cellular signaling pathways. For exogenous viruses, this tweaking of cellular function serves to enhance their replicative success through the modulation of pathways related to virion production, dissemination, cell survival, and immunity [1,2]. It is less clear in what manner ever-present viral symbionts such as endogenous retroviruses (ERVs) interact with the proteome of their hosts.

The genomes of eukaryotic organisms are widely populated with ERVs [3,4,5]. Endogenous retrovirus-K (ERVK/HERV-K) is a biomedically-relevant symbiont within the primate lineage [6,7]. Its expression has been associated with a variety of cancers [8], neurological conditions [9,10,11,12,13], autoimmune diseases [14], and infections [13,15]. A common thread that weaves through ERVK-associated disease is genomic instability. DNA damage and genomic alterations are hallmarks of many cancers [16], as well as in the neurons of patients with the motor neuron disease ALS [17,18]. One protein known to cause DNA damage during the retroviral life cycle is the integrase (IN) enzyme [19]. Recovery from IN-driven lesions is reliant on the host DNA damage response (DDR) [20,21].

We have previously shown that several ERVK insertions in the human genome have the potential to produce functional ERVK IN enzymes with the identical DDE active site motif found in human immunodeficiency virus (HIV) IN [22]. Based on homology modeling, we predict that the ERVK IN enzyme contains all the essential motifs and domain structures for retroviral IN function [22]. A recombinant ERVK-10 integrase enzyme also confirms that it has the potential for strand-transfer activity [23]. A remaining question is how ERVK IN interacts with cellular proteins and pathways, as has been shown for many other retroviral integrases [19,24,25,26].

Retroviral INs are involved in pre-integration complex (PIC) transport [27], viral genome integration into host DNA [19], and virion maturation [28]. Thus, retroviral integrase enzymes exhibit a diversity of cellular partners and have been shown to impact cell signaling and survival processes, including the DDR [19,29]. For example, retroviral IN often recruits viral proteins (reverse transcriptase, matrix, and capsid) and cellular factors (BANF1, HGMA1, LEDGF) to participate in the viral DNA integration process [30,31]. Moreover, successful viral DNA integration requires engagement of the host DDR proteins to repair residual single-stranded DNA gaps flanking the integration site [20,29,32]. In contrast, failed provirus insertion or unresolved lesions can lead to double-stranded DNA (dsDNA) breaks in the host genome [29]. The level of γH2AX foci is positively correlated with the number of double-stranded DNA breaks (DSB) in mammalian cells, and it is widely used as a quantitative biomarker of retrovirus-mediated DSBs [19,33,34]. This genomic damage is particularly hazardous to the cell, as DSB potentially lead to chromosomal rearrangements, cellular deregulation, and apoptosis [35,36]. Thus, as an intrinsic protective measure, select host proteins (RAD51, Kap1, TREX1, p21, HDAC10, TRIM33) are known antagonists of the retroviral integration process [31,37,38,39,40]. Many studies have identified direct protein binding partners and cellular complexes which interact with HIV integrase [41,42,43]; in contrast, the ERVK IN interactome remains unknown.

A complicating factor for the development of model systems to study the impact of ERVK proteins in vivo is that many other organisms contain ERVs with similarity to ERVK. Given the known cellular impacts of retroviral integrases, we hypothesized that a computational biology approach would identify potential cellular partners of ERVK IN and point toward its capacity to modulate cellular pathways. Additionally, a comparison with similar integrases in eukaryotic organism and model species may inform the future establishment of in vivo models for ERVK IN-driven pathology.

2. Materials and Methods

2.1. Database Curation

Integrases with sequence similarity to ERVK IN (based on ERVK-10 [22]; P10266.2) were identified using the National Centre for Biotechnology’s (NCBI) Protein–protein Basic Local Alignment Search Tool (BLASTp) within the non-redundant (nr), model organisms (mo), and transcriptome shotgun assembly proteins (tsa) databases [44]. Default algorithm parameters were used, with E-value cut-offs for each database as follows: E < 3.0 × 10⁻⁷⁰ (nr), E < 0.01 (mo), E < 2.0 × 10⁻¹⁰ (tsa). Sequences were grouped based on phylogeny as informed by ICTV (International Committee on Taxonomy of Viruses; 2021, https://talk.ictvonline.org/ (accessed on 16 May 2021)) or OneZoom (OneZoom Tree of Life Explorer; version 3.4.1; Software for Technical Computation; United Kingdom, 2021, https://www.onezoom.org/ (accessed on 16 May 2021)) [45] and are listed in

Table A1. Exogenous viruses with similarity to ERVK integrase based on BLASTp search.

Host	Species	Protein Name	Accession Number	Database	Percent Identity	E Value	Alignment
Retroviridae—Betaretrovirus
Mus Musculus	Mouse mammary tumor virus (MMTV)	MMTV strain BR6 Integrase, partial	5CZ2_A	nr	56.46%	3.00 × 10−71
		Unnamed protein product	CAA25954.1	nr	52.36%	2.00 × 10−70
		Chain A. Integrase (Core model of the MMTV Intasome)	3JCA_A	nr	51.50%	2.00 × 10−82
		Pr160	NP_056880.1	nr	51.11%	7.00 × 10−76
		Pr160 gag pro pol precursor	AAA46542.1	nr	51.11%	7.00 × 10−76
		p30DU-p13PR-RT-IN	NP_955564.1	nr	51.11%	2.00 × 10−76
		Gag-Pro-Pol polyprotein	P11283.2	nr	51.11%	3.00 × 10−75
		MMTV putative integrase	AAC24859.1	nr	50.37%	5.00 × 10−81
		Gag-Pol-Pro polyprotein, partial	BAA03767.1	nr	49.65%	1.00 × 10−77
		Exogenous MMTV Gag-Pro-Pol	AAF31469.1	nr	49.65%	4.00 × 10−76	•
		Endogenous MTV1 Gag-Pro-Pol	AAF31464.1	nr	49.65%	4.00 × 10−76
Capra hircus	Enzootic Nasal Tumor Virus (ENTV)	Pol protein, partial	AVG72436.1	nr	48.79%	3.00 × 10−72
		Pol protein, partial	AVG72437.1	nr	48.79%	3.00 × 10−71
		Pol protein, partial	AVG72438.1	nr	48.79%	7.00 × 10−71
		Pol protein, partial	AVG72441.1	nr	48.39%	3.00 × 10−71
		Pol protein, partial	AVG72440.1	nr	48.39%	4.00 × 10−71
		Pol protein, partial	AVG72435.1	nr	48.39%	2.00 × 10−70
		Gag-Pro-Pol protein	QBP05340.1	nr	47.41%	3.00 × 10−73
		Pol protein, partial	ANG58667.1	nr	47.41%	2.00 × 10−72
		Pol protein	QEQ26602.1	nr	47.41%	3.00 × 10−72
		Pol protein, partial	ANG58699.1	nr	47.41%	2.00 × 10−71	•
		Pol protein, partial	ANG58695.1	nr	47.41%	2.00 × 10−71
		Pol protein, partial	ANG58691.1	nr	47.41%	2.00 × 10−71
		Pol protein, partial	ANG58679.1	nr	47.41%	5.00 × 10−71
		Endogenous ENTV pol protein	QPG92760.1	nr	47.41%	3.00 × 10−73
		Endogenous ENTV pol protein	QPG92768.1	nr	47.41%	1.00 × 10−72
		Gag-Pro-Pol, partial	AOZ60515.1	nr	47.04%	1.00 × 10−72
		Pol protein, partial	ANG58671.1	nr	47.04%	1.00 × 10−71
		Pol protein, partial	ANG58683.1	nr	47.04%	3.00 × 10−70
		Pol protein, partial	ANG58687.1	nr	47.04%	2.00 × 10−71
		Pol protein, partial	ANG58663.1	nr	47.04%	8.00 × 10−71
		Gag-Pro-Pol protein	ANC55859.1	nr	46.67%	5.00 × 10−73
		Gag-Pro-Pol protein, partial	ADI50273.1	nr	46.67%	4.00 × 10−72
		Gag-Pro-Pol protein, partial	AOZ60519.1	nr	46.67%	7.00 × 10−72
		Pol protein, partial	ANG58675.1	nr	46.67%	3.00 × 10−71
		Gag-Pro-Pol fusion, partial	NP_862833.2	nr	46.67%	7.00 × 10−71
		Pol protein, partial	ANG58659.1	nr	46.10%	1.00 × 10−72
Macaca genus	Mason-Pfizer monkey virus (M-PMV)	Simian AIDS retrovirus SRV-1-Pol polyprotein	GNLJSA	nr	48.30%	2.00 × 10−70
		Simian retrovirus SRV-5-Pol polyprotein	BBG56792.1	nr	47.57%	5.00 × 10−71	•
		Simian retrovirus SRV-Y-Pol protein, partial	BAM71050.1	nr	47.57%	2.00 × 10−70
Ovis Aries	Jaagsiekte sheep retrovirus	Reverse transcriptase, partial	CAA77117.1	nr	46.67%	5.00 × 10−75
		Reverse transcriptase, partial	CAA77113.1	nr	46.67%	2.00 × 10−74
		Pol protein	NP_041186.1	nr	46.30%	1.00 × 10−70	•

BLAST databases: nr denotes non-redundant protein database. Circle symbol: denotes integrase sequences used in protein alignment in Figure 1.

,

Table A2. Boreoeutherian genomes with similarity to ERVK integrase based on BLASTp searches.

Species		Protein Name	Accession Number	Database	Percent Identity	E Value
Homo sapiens	Human Endogenous Retrovirus K (HERV-K/ ERVK)	Pol/env ORF (bases 3878-8257) first start codon at 4172; Xxx; putative	AAA88033.1	nr	100.00%	0
		Endogenous retrovirus group K member 10 Pol protein	P10266.2	nr	100.00%	0
		Gag-Pro-Pol-Env protein	AAD51793.1	nr	100.00%	0
		Pol protein, partial	CAA71417.1	nr	100.00%	2.00 × 10−101
		Endogenous retrovirus group K member 11 Pol protein	Q9UQG0.2	nr	99.64%	0
		Endogenous retrovirus group K member 7 Pol protein	P63135.1	nr	99.29%	0
		Reverse transcriptase, partial	AGQ55918.1	nr	99.28%	1.00 × 10−94
		Reverse transcriptase, partial	AGQ55922.1	nr	99.28%	7.00 × 10−94
		Polymerase, partial	AAO27434.1	nr	99.27%	0
		Gag-Pro-Pol protein	AAD51796.1	nr	99.17%	5.00 × 10−161
		Endogenous retrovirus group K member 113 Pol protein	P63132.1	nr	98.21%	0
		Polymerase, partial	AAK11553.1	nr	98.21%	0
		Endogenous retrovirus group K member 6 Pol protein	Q9BXR3.2	nr	98.21%	0
		Endogenous retrovirus group K member 8 Pol protein	P63133.1	nr	98.21%	0
		Gag-Pro-Pol protein	AAD51797.1	nr	98.21%	0
		Pol protein	CAA76885.1	nr	97.86%	0
		Pol protein	CAA76879.1	nr	97.86%	0
		Polymerase, partial	AAK11554.1	nr	97.86%	0
		Reverse transcriptase, partial	AGQ55928.1	nr	97.84%	6.00 × 10−93
		Reverse transcriptase, partial	AGQ55923.1	nr	97.84%	8.00 × 10−93
		Reverse transcriptase, partial	AGQ55925.1	nr	97.83%	2.00 × 10−92
		Reverse transcriptase, partial	AGQ55927.1	nr	97.83%	3.00 × 10−92
		Reverse transcriptase, partial	AGQ55914.1	nr	97.76%	6.00 × 10−89
		Pol protein	CAA76882.1	nr	97.50%	0
		Endogenous retrovirus group K member 19 Pol protein	Q9WJR5.2	nr	97.50%	0
		Endogenous retrovirus group K member 25 Pol protein	P63136.1	nr	97.50%	0
		Pol protein	AAL60056.1	nr	97.30%	1.00 × 10−152
		Reverse transcriptase, partial	AGQ55924.1	nr	97.12%	2.00 × 10−92
		Reverse transcriptase, partial	AGQ55926.1	nr	97.10%	9.00 × 10−92
		Reverse transcriptase, partial	AGQ55921.1	nr	97.10%	9.00 × 10−92
		Reverse transcriptase, partial	AGQ55919.1	nr	97.10%	1.00 × 10−91
		Pol/env protein, partial	AET81039.1	nr	96.97%	1.00 × 10−81
		Reverse transcriptase, partial	AGQ55920.1	nr	96.38%	2.00 × 10−91
		Pol protein	CAB56603.1	nr	90.13%	2.00 × 10−132
		Endogenous retrovirus group K member 18 Pol protein	Q9QC07.2	nr	90.13%	2.00 × 10−130
		hCG1808534	EAW92672.1	nr	87.38%	3.00 × 10−130
Macaca fascicularis		PREDICTED: endogenous retrovirus group K member 8 Pol protein-like	XP_015309771.1	nr	83.77%	2.00 × 10−127
Chlorocebus sabaeus		Pol protein, partial	BBC20786.1	nr	47.01%	3.00 × 10−70
Oryctolagus cuniculus		PREDICTED: endogenous retrovirus group K member 8 Pol protein-like	XP_017205812.1	nr	49.82%	1.00 × 10−76
Marmota marmota		PREDICTED: endogenous retrovirus group K member 11 Pol protein-like	XP_015349278.1	nr	47.96%	4.00 × 10−71
Ochotona princeps		Uncharacterized protein LOC105942652	XP_012786727.1	nr	47.33%	1.00 × 10−70
Mus musculus (mouse)		Agouti-signaling protein isoform X1	XP_017174599.2	mo	46.93%	6.00 × 10−70
		Contactin-5 isoform X2	XP_036010832.1	mo	44.11%	3.00 × 10−63
		Contactin-5 isoform X1	XP_036010831.1	mo	44.11%	4.00 × 10−63
		Contactin-5 isoform X3	XP_036010833.1	mo	44.11%	4.00 × 10−63
		Protein NYNRIN-like isoform X1	XP_036020530.1	mo	30.15%	9.00 × 10−14
		Uncharacterized protein Gm39701	XP_011237194.2	mo	30.15%	9.00 × 10−14
		Uncharacterized protein LOC118567641, partial	XP_036010828.1	mo	29.71%	2.00 × 10−12
Fukomys damarensis		Pol polyprotein	KFO35018.1	nr	45.14%	1.00 × 10−71
Sus scrofa		TPA: uncharacterized protein	HCZ91355.1	tsa	61.54%	2.00 × 10−65
		TPA: uncharacterized protein	HDB33152.1	tsa	61.54%	2.00 × 10−65
		Endogenous retrovirus group K member 11 Pol protein-like	HDB62800.1	tsa	60.66%	2.00 × 10−17
		TPA: uncharacterized protein	HCZ89879.1	tsa	52.24%	1.00 × 10−39
		Nuclear autoantigenic sperm protein isoform X4	HDA97069.1	tsa	49.06%	2.00 × 10−21
		Nuclear autoantigenic sperm protein isoform 2	HCZ78574.1	tsa	48.86%	5.00 × 10−15
		Nuclear autoantigenic sperm protein isoform 2	HDB80991.1	tsa	48.42%	7.00 × 10−17
		TPA: uncharacterized protein	HCZ87894.1	tsa	42.67%	5.00 × 10−58
		TPA: uncharacterized protein	HDB20633.1	tsa	41.78%	1.00 × 10−53
		TPA: uncharacterized protein	HDC25054.1	tsa	39.81%	7.00 × 10−48
		TPA: uncharacterized protein	HDA81201.1	tsa	38.84%	1.00 × 10−47
		Transmembrane protein 161B isoform X6-like	HDB13612.1	tsa	31.52%	2.00 × 10−13
		TPA: uncharacterized protein	HDC69173.1	tsa	31.07%	2.00 × 10−13
		TPA: uncharacterized protein	HDC69046.1	tsa	30.90%	3.00 × 10−13
		Transmembrane protein 161B isoform X12-like	HDC79805.1	tsa	30.81%	1.00 × 10−12
		Transmembrane protein 161B isoform X12-like	HDB85007.1	tsa	30.81%	1.00 × 10−12
		Transmembrane protein 161B isoform X6-like	HDA96697.1	tsa	30.81%	1.00 × 10−12
		Transmembrane protein 161B isoform X12-like	HDB79678.1	tsa	30.81%	2.00 × 10−12
		Transmembrane protein 161B isoform X6	HDB81123.1	tsa	30.77%	1.00 × 10−11
		Transmembrane protein 161B isoform X6	HDB82421.1	tsa	30.71%	2.00 × 10−10
		TPA: uncharacterized protein	HDC70342.1	tsa	30.41%	6.00 × 10−12
		TPA: uncharacterized protein	HDC70174.1	tsa	30.41%	6.00 × 10−12
		TPA: uncharacterized protein	HDC69016.1	tsa	30.41%	6.00 × 10−12
		putative protein-like	HDB95244.1	tsa	30.23%	3.00 × 10−12
		TPA: uncharacterized protein	HDB49716.1	tsa	30.23%	5.00 × 10−12
		TPA: uncharacterized protein	HDB49718.1	tsa	30.23%	5.00 × 10−12
		TPA: uncharacterized protein	HDC70066.1	tsa	30.23%	5.00 × 10−12
		TPA: uncharacterized protein	HDC69012.1	tsa	30.23%	7.00 × 10−12
		TPA: uncharacterized protein	HDB50090.1	tsa	29.82%	2.00 × 10−11
		TPA: uncharacterized protein	HDB54298.1	tsa	29.65%	1.00 × 10−11
		Transmembrane protein 161B isoform X3-like	HDC79806.1	tsa	29.65%	2.00 × 10−11
		Endogenous retrovirus group K member 25 Pol	HCZ79815.1	tsa	29.65%	3.00 × 10−11
		Transmembrane protein 161B isoform X2-like	HDC79761.1	tsa	29.59%	1.00 × 10−10
		Endogenous retrovirus group K member 25 Pol	HDA78544.1	tsa	29.24%	4.00 × 10−11
		Endogenous retrovirus group K member 25 Pol	HDA79987.1	tsa	29.24%	7.00 × 10−11
		TPA: uncharacterized protein	HDA79350.1	tsa	29.24%	9.00 × 10−11
		Endogenous retrovirus group K member 25 Pol	HDA79988.1	tsa	29.24%	9.00 × 10−11
		TPA: uncharacterized protein	HDA79349.1	tsa	29.24%	1.00 × 10−10
		TPA: uncharacterized protein	HDC13198.1	tsa	29.24%	1.00 × 10−10
		Transmembrane protein 161B isoform X6-like	HDB88647.1	tsa	29.07%	3.00 × 10−11
		TPA: uncharacterized protein	HDA61679.1	tsa	29.07%	5.00 × 10−11
		TPA: uncharacterized protein	HDB82700.1	tsa	29.07%	1.00 × 10−10
Equus asinus		PREDICTED: endogenous retrovirus group K member 8 Pol protein-like	XP_014715024.1	nr	56.46%	3.00 × 10−95
Capra hircus		PREDICTED: LOW QUALITY PROTEIN: endogenous retrovirus group K member 18 Pol protein-like	XP_017905435.1	nr	47.41%	3.00 × 10−71
Ovis aries		Pol protein	ABV71120.1	nr	47.04%	2.00 × 10−72
		Pol protein	ABV71104.1	nr	46.67%	4.00 × 10−71
		Pol protein	ABV71084.1	nr	46.67%	4.00 × 10−71
		Pol protein	ABV71074.1	nr	46.67%	4.00 × 10−71
		Pol protein	ABV71079.1	nr	46.67%	4.00 × 10−71
		Pol protein	ABV71069.1	nr	46.67%	4.00 × 10−71
		Pol protein (endogenous virus)	AST51848.1	nr	46.67%	5.00 × 10−71
		Pol protein	ABV71094.1	nr	46.30%	2.00 × 10−70

BLAST databases: nr = non-redundant protein database; mo = model organisms database; tsa = transcriptomics shotgun analysis non-redundant database. TPA = third party annotation.

,

Table A3. Euteleostomi genomes with similarity to ERVK integrase based on BLASTp searches.

Species	Protein Name	Accession Number	Data base	Percent Identity	E Value
Micrurus lemniscatus carvalhoi	Hypothetical protein, partial	LAA32932.1	tsa	58.47%	2.00 × 10−40
	Hypothetical protein, partial	LAA32929.1	tsa	57.63%	6.00 × 10−40
	Hypothetical protein, partial	LAA32939.1	tsa	48.29%	4.00 × 10−54
	Hypothetical protein, partial	LAA32941.1	tsa	44.00%	1.00 × 10−26
Zosterops borbonicus	Hypothetical protein HGM15179_011615	TRZ15504.1	nr	46.97%	3.00 × 10−71
Zonotrichia albicollis	Uncharacterized protein LOC106629581	XP_014125095.1	nr	46.24%	1.00 × 10−71
Micrurus corallinus	Hypothetical protein, partial	LAA64555.1	tsa	46.21%	2.00 × 10−26
	Hypothetical protein, partial	LAA64556.1	tsa	45.08%	2.00 × 10−21
Micrurus lemniscatus lemniscatus	Hypothetical protein, partial	LAA89554.1	tsa	44.57%	4.00 × 10−18
	Hypothetical protein, partial	LAA89545.1	tsa	43.75%	1.00 × 10−23
Bird metagenome	Gag-Pro-Pol polyprotein, partial	MBY11728.1	tsa	44.31%	3.00 × 10−43
Gallirallus okinawae	Hypothetical protein, partial	LAC45429.1	tsa	43.96%	3.00 × 10−57
Fundulus heteroclitus	Integrase core domain, partial	JAQ81073.1	tsa	35.05%	2.00 × 10−11
Danio rerio (zebrafish)	Uncharacterized protein LOC108190699	XP_017212567.1	mo	33.64%	4.00 × 10−12
	Uncharacterized protein K02A2.6-like	XP_021334762.1	mo	30.18%	4.00 × 10−11
	Uncharacterized protein K02A2.6-like	XP_017210639.2	mo	29.20%	8.00 × 10−9
	Uncharacterized protein LOC101886116	XP_021327301.1	mo	28.47%	1.00 × 10−5
	Uncharacterized protein LOC110439859	XP_021332670.1	mo	28.17%	0.001
	Uncharacterized protein K02A2.6-like	XP_003199161.1	mo	27.78%	5.00 × 10−10
	Uncharacterized protein K02A2.6-like	XP_002663225.3	mo	27.78%	8.00 × 10−10
	Uncharacterized protein LOC110438047	XP_021323131.1	mo	26.43%	0.006
Nothobranchius korthausae	Uncharacterized protein	SBQ67355.1	tsa	32.02%	8.00 × 10−11
Nothobranchius kadleci	Uncharacterized protein	SBP84572.1	tsa	32.00%	8.00 × 10−11
Nothobranchius furzeri	Uncharacterized protein	SBS54329.1	tsa	31.25%	5.00 × 10−13
Nothobranchius rachovii	Uncharacterized protein	SBS11479.1	tsa	30.07%	2.00 × 10−10

BLAST databases: nr = non-redundant protein database; mo = model organisms database; tsa = transcriptomics shotgun analysis non-redundant database.

and

Table A4. Nephrozoa (non-boreoeutherian) genomes with similarity to ERVK integrase based on BLASTp searches.

Species	Protein Name	Accession Number	Data base	Percent Identity	E Value
Onchocerca flexuosa	Integrase core domain protein	OZC05619.1	nr	45.68%	6.00 × 10−73
Ixodes ricinus	Putative tf2-11 polyprotein	MXV00662.1	tsa	35.81%	2.00 × 10−10
	Putative tick transposon, partial	JAP73380.1	tsa	30.67%	1.00 × 10−11
	Putative tick transposon, partial	JAR90689.1	tsa	30.67%	6.00 × 10−11
	Putative bell all, partial	JAA73039.1	tsa	30.00%	2.00 × 10−10
	Putative gypsy11-i sp, partial	JAP69562.1	tsa	30.00%	2.00 × 10−10
	Putative tick transposon, partial	JAR92200.1	tsa	28.06%	4.00 × 10−13
	Putative transposon tf2-9 polyprotein	MXV00940.1	tsa	27.37%	9.00 × 10−13
Littorina littorea	Transposon Ty3-G Gag-Pol polyprotein	MBX96210.1	tsa	35.54%	4.00 × 10−12
	Transposon Ty3-I Gag-Pol polyprotein	MBX97975.1	tsa	30.00%	4.00 × 10−14
Ixodes scapularis	Putative tick transposon, partial	MOY42200.1	tsa	34.29%	4.00 × 10−12
	Putative tick transposon, partial	MOY42203.1	tsa	32.67%	2.00 × 10−13
	Putative tick transposon, partial	MOY42202.1	tsa	32.67%	7.00 × 10−13
	Putative gypsy-17 ga-i, partial	MOY42236.1	tsa	31.09%	2.00 × 10−12
	Hypothetical protein, partial	MOY42219.1	tsa	30.00%	6.00 × 10−11
	Putative gypsy11-i sp, partial	MOY42209.1	tsa	29.17%	7.00 × 10−12
	Putative gypsy21-i sp, partial	MOY42223.1	tsa	29.17%	8.00 × 10−12
	Putative tick transposon, partial	MOY42227.1	tsa	28.50%	4.00 × 10−13
	Putative tick transposon, partial	MOY42214.1	tsa	28.25%	9.00 × 10−11
	Putative gypsy-27 xt-i, partial	MOY35588.1	tsa	26.44%	1.00 × 10−10
Lygus hesperus	Retrotransposable element Tf2 protein type 3, partial	JAQ12551.1	tsa	33.58%	6.00 × 10−13
	Uncharacterized protein	JAG20083.1	tsa	33.58%	6.00 × 10−13
	Hypothetical protein, partial CM83_13537, partial	JAG31779.1	tsa	29.93%	5.00 × 10−11
	Uncharacterized protein, partial K02A2.6, partial	JAG32119.1	tsa	28.87%	1.00 × 10−11
	Uncharacterized protein, partial K02A2.6, partial	JAG38901.1	tsa	26.82%	8.00 × 10−12
Amblyomma sculptum	Putative gypsy-7 adi-i, partial	JAU00762.1	tsa	32.88%	1.00 × 10−12
	Putative tick transposon, partial	JAU00733.1	tsa	30.00%	2.00 × 10−10
	Putative tick transposon, partial	JAU00744.1	tsa	30.00%	2.00 × 10−10
Rhipicephalus microplus	Putative tick transposon	NIE47479.1	tsa	32.45%	4.00 × 10−11
Strongylocentrotus purpuratus	Integrase, catalytic core containing protein	MOS08875.1	tsa	32.19%	2.00 × 10−16
	Integrase, catalytic core containing protein	MOS08729.1	tsa	32.03%	7.00 × 10−13
	Integrase, catalytic core containing protein	MOS14051.1	tsa	30.57%	2.00 × 10−13
	Reverse transcriptase domain-containing protein	MOS08491.1	tsa	30.57%	2.00 × 10−13
	Reverse transcriptase domain-containing protein	MOS11069.1	tsa	29.93%	5.00 × 10−12
	Integrase, catalytic core containing protein	MOS08728.1	tsa	29.76%	2.00 × 10−10
	Integrase, catalytic core containing protein	MOS08842.1	tsa	28.57%	4.00 × 10−11
	Integrase, catalytic core containing protein	MOS08745.1	tsa	24.12%	2.00 × 10−10
Ornithodoros turicata	Putative transposon tf2-9 polyprotein	MBY06492.1	tsa	32.00%	1.00 × 10−10
Ornithodoros moubata	Esterase D, partial	JAW02195.1	tsa	29.86%	2.00 × 10−10
Photinus pyralis	Hypothetical protein	JAV53763.1	tsa	29.20%	2.00 × 10−13
Lepeophtheirus salmonis	Uncharacterized protein K02A2.6like, partial	CDW28658.1	tsa	28.28%	1.00 × 10−10
Drosophila melanogaster (Fruit fly)	Unnamed protein product	CAA30503.1	nr-DM	28.87%	0.004
	Sd02026p	AAK84933.1	nr-DM	28.57%	3.00 × 10−9
	Blastopia polyprotein	CAA81643.1	nr-DM	28.19%	2.00 × 10−9
	Polyprotein	ACI62137.1	nr-DM	28.00%	2.00 × 10−4

BLAST databases: nr = non-redundant protein database; nr-DM = non-redundant protein database with Drosophila specified as species search constraint; tsa = transcriptomics shotgun analysis non-redundant database.

.

2.2. Protein Alignments and Eukaryotic Linear Motif Annotation

The ERVK IN protein sequence, as well as select representative integrases from exogenous Betaretroviruses (Figure 1) or endogenous retroviruses (Figure 2) were aligned using Geneious Prime (version 2021.0.3; Software for Technical Computation; San Diego, CA, USA; Auckland, New Zealand, 2021) software [46]. A global alignment with free end gaps using BLOSUM62 matrix was performed. Longer sequences were truncated to overlap with the ERVK IN reference sequence. Figures depict the sequence logo and integrase active sites, with HHCC and DDE regions highlighted based on Conserved Domains Database (CDD) annotation [47].

Each aligned integrase sequence was submitted to the Eukaryotic Linear Motif (ELM; Software for Technical Computation; 2020, http://elm.eu.org/ (accessed on 16 May 2021)) resource [48]. A complete listing of ELMs identified in each integrase is presented in

Table 1. ELM motifs in integrases from ERVK and exogenous betaretroviruses.

ELM Motif		ELM Accession	Alignment Notation	Integrase					Conservation
				ERVK	MMTV	M-PMV	ENTV	JSRV
Cleavage and degradation	CLV_C14_Caspase3-7	ELME000321		1	0	0	0	2	0.4
	CLV_PCSK_KEX2_1	ELME000108		1	0	0	0	0	0.2
	CLV_NRD_NRD_1	ELME000102		0	1	0	1	1	0.6
	CLV_PCSK_PC1ET2_1	ELME000100		1	0	0	0	0	0.2
	CLV_PCSK_SKI1_1	ELME000146		5	4	4	1	0	0.8
	DEG_APCC_DBOX_1	ELME000231		0	0	0	1	0	0.2
Docking	DOC_CKS1_1	ELME000358		0	1	0	0	0	0.2
	DOC_CYCLIN_RxL_1	ELME000106	Cyclin	2	0	2	0	0	0.4
	DOC_MAPK_gen_1	ELME000233		0	2	0	1	1	0.6
	DOC_MAPK_MEF2A_6	ELME000432	ERK/p38	1	1	0	1	1	0.8
	DOC_PP1_RVXF_1	ELME000137	PP1c	3	1	1	1	1	1.0
	DOC_PP2B_LxvP_1	ELME000367		1	0	1	0	0	0.4
	DOC_PP4_FxxP_1	ELME000477		0	1	0	1	1	0.6
	DOC_USP7_MATH_1	ELME000239	USP7_M	1	3	0	2	2	0.8
	DOC_USP7_UBL2_3	ELME000394	USP7_U	1	0	1	0	0	0.4
	DOC_WW_Pin1_4	ELME000136	Pin1	3	6	1	3	2	1.0
Ligand	LIG_14-3-3_CanoR_1	ELME000417	14-3-3	2	1	1	2	2	1.0
	LIG_14-3-3_CterR_2	ELME000418	14-3-3 *	1	0	0	0	0	0.2
	LIG_Actin_WH2_2	ELME000313		0	1	0	1	1	0.6
	LIG_APCC_ABBA_1	ELME000435		0	1	0	0	0	0.2
	LIG_BIR_II_1	ELME000285	IAP	1	1	1	1	1	1.0
	LIG_BIR_III_4	ELME000293		0	0	0	1	0	0.2
	LIG_BRCT_BRCA1_1	ELME000197	BRCT	1	0	1	1	1	0.8
	LIG_BRCT_BRCA1_2	ELME000198	BRCT1 *	1	0	0	0	0	0.2
	LIG_CSL_BTD_1	ELME000410		1	1	0	0	0	0.4
	LIG_EH1_1	ELME000148		0	0	1	0	1	0.4
	LIG_eIF4E_1	ELME000317		0	1	0	0	0	0.2
	LIG_FHA_1	ELME000052	FHA	5	1	2	0	1	0.8
	LIG_FHA_2	ELME000220	FHA 2	1	1	1	0	0	0.6
	LIG_LIR_Apic_2	ELME000369		0	3	2	1	1	0.8
	LIG_LIR_Gen_1	ELME000368	LIR	2	0	0	0	0	0.2
	LIG_MAD2	ELME000167		0	0	0	1	0	0.2
	LIG_NRBOX	ELME000045		0	0	0	1	0	0.2
	LIG_LIR_Nem_3	ELME000370		0	6	7	1	4	0.8
	LIG_Pex14_1	ELME000080	Pex14	1	0	0	0	0	0.2
	LIG_Pex14_2	ELME000328	Pex14	2	2	1	1	1	1.0
	LIG_PTB_Apo_2	ELME000122		0	0	1	0	1	0.4
	LIG_PTB_Phospho_1	ELME000095		0	0	0	0	1	0.2
	LIG_RPA_C_Fungi	ELME000382		0	0	1	1	1	0.6
	LIG_SH2_CRK	ELME000458		0	2	0	0	0	0.2
	LIG_SH2_NCK_1	ELME000474		0	1	0	0	0	0.2
	LIG_SH2_PTP2	ELME000083	SH2	1	1	0	1	1	0.8
	LIG_SH2_SRC	ELME000081	SH2	1	1	1	1	1	1.0
	LIG_SH2_STAP1	ELME000465		2	1	0	0	0	0.4
	LIG_SH2_STAT3	ELME000163	STAT3	2	1	1	1	1	1.0
	LIG_SH2_STAT5	ELME000182	STAT5	3	3	2	3	3	1.0
	LIG_SH3_1	ELME000005		0	1	0	0	0	0.2
	LIG_SH3_3	ELME000155	SH3	1	2	1	1	1	1.0
	LIG_SH3_4	ELME000156		0	0	0	1	0	0.2
	LIG_SxIP_EBH_1	ELME000254	EBH	1	1	1	1	2	1.0
	LIG_TRAF2_1	ELME000117	TRAF2	1	1	0	1	1	0.8
	LIG_TYR_ITIM	ELME000020	ITIM	1	1	1	1	1	1.0
	LIG_Vh1_VBS_1	ELME000438		0	0	0	0	1	0.2
	LIG_WD40_WDR5_VDV_2	ELME000365	WDR5	2	10	3	8	9	1.0
	LIG_WW_3	ELME000135		0	1	0	0	0	0.2
Modification	MOD_CDK_SPK_2	ELME000429		0	0	1	0	0	0.2
	MOD_CDK_SPxK_1	ELME000153		0	1	0	0	0	0.2
	MOD_CK1_1	ELME000063	CK1-P	1	3	2	4	4	1.0
	MOD_CK2_1	ELME000064		3	3	1	0	0	0.6
	MOD_Cter_Amidation	ELME000093		1	0	0	0	0	0.2
	MOD_GlcNHglycan	ELME000085	GlyNH	1	2	3	1	2	1.0
	MOD_GSK3_1	ELME000053	GSK3-P	3	4	2	1	2	1.0
	MOD_NEK2_1	ELME000336	NEK2-P	2	3	3	2	4	1.0
	MOD_NEK2_2	ELME000337		0	0	1	0	0	0.2
	MOD_N-GLC_1	ELME000070		1	0	1	2	0	0.6
	MOD_PIKK_1	ELME000202		5	0	1	0	0	0.4
	MOD_PKA_1	ELME000008		0	1	0	0	0	0.2
	MOD_PKA_2	ELME000062	PKA-P	1	0	1	2	2	0.8
	MOD_Plk_1	ELME000442	PLK-P	4	1	2	1	1	1.0
	MOD_Plk_4	ELME000444		1	0	1	0	0	0.4
	MOD_ProDKin_1	ELME000159	p38-P	3	6	1	3	2	1.0
	MOD_SUMO_rev_2	ELME000393		1	1	0	0	0	0.4
Target	TRG_ENDOCYTIC_2	ELME000120		2	4	2	1	1	1.0
	TRG_Pf-PMV_PEXEL_1	ELME000462		1	2	1	1	1	1.0

GenBank accession numbers for betaretroviral integrase sequences are as follows: Endogenous retrovirus-K (ERVK; P10266.2), Exogenous mouse mammary tumor virus (MMTV; AAF31469.1), Mason–Pzifer monkey virus 5 (M-PMV; BBG56792.1), Enzootic nasal tumor virus (ENTV; ANG58699.1) and Jaagsiekte sheep retrovirus (JSRV; NP_041186.1). Asterisk indicates ERVK-specific ELM motif in Figure 1.

and

Table 2. ELM motifs in ERVK integrase and similar endogenous integrases in eukaryotes.

ELM Motif		ELM Accession	ERVK Integrase (Homo sapiens)	ERVK-8 Pol protein-Like (Macaca fascicularis)	Pol Protein (Chlorocebus sabaeus)	Pol Protein (Fukomys darmarensis)	Putative Protein (Ochonta princeps)	ERVK-8 pol Protein-Like (Equus asinus)	ERVK-18 pol Protein-Like (Capra hircus)	Pol Protein (Ovis aries)	Putative Protein (Zonotrichia albicollis)	Putative Protein (Zosterops borbonicus)	Integrase (Onchocerca flexuosa)	Motif conservation
Cleavage	CLV_C14_Caspase3-7	ELME000321	1	0	0	0	1	0	0	0	0	0	0	0.2
	CLV_NRD_NRD_1	ELME000102	0	0	0	0	0	0	0	1	0	0	1	0.2
	CLV_PCSK_KEX2_1	ELME000108	1	2	0	0	0	0	0	0	0	1	0	0.3
	CLV_PCSK_PC1ET2_1	ELME000100	1	2	0	0	0	0	0	0	0	1	0	0.3
	CLV_PCSK_SKI1_1	ELME000146	5	3	4	3	7	8	0	0	3	5	2	0.8
Degradation	DEG_APCC_DBOX_1	ELME000231	0	0	0	0	0	0	0	0	0	0	1	0.1
	DEG_MDM2_SWIB_1	ELME000184	0	0	0	0	0	0	0	0	1	0	0	0.1
Docking	DOC_CKS1_1	ELME000358	0	1	0	0	2	0	0	0	1	2	1	0.5
	DOC_CYCLIN_RxL_1	ELME000106	2	1	2	0	2	2	0	0	3	3	1	0.7
	DOC_MAPK_DCC_7	ELME000433	0	0	0	1	0	0	0	0	0	0	1	0.2
	DOC_MAPK_gen_1	ELME000233	0	1	0	3	5	2	1	1	2	4	1	0.8
	DOC_MAPK_HePTP_8	ELME000434	0	0	0	0	0	0	0	0	0	0	1	0.1
	DOC_MAPK_MEF2A_6	ELME000432	1	1	0	0	2	1	1	1	3	4	3	0.8
	DOC_PP1_RVXF_1	ELME000137	3	3	1	1	0	0	1	1	0	1	1	0.7
	DOC_PP2A_B56_1	ELME000425	0	0	0	1	0	0	0	0	0	0	0	0.1
	DOC_PP2B_LxvP_1	ELME000367	1	1	2	0	0	0	0	0	1	1	1	0.5
	DOC_PP2B_PxIxI_1	ELME000237	0	0	0	0	1	0	0	0	0	0	0	0.1
	DOC_PP4_FxxP_1	ELME000477	0	1	1	1	1	1	1	0	0	0	2	0.6
	DOC_USP7_MATH_1	ELME000239	1	3	0	1	1	1	2	2	3	1	3	0.9
	DOC_USP7_MATH_2	ELME000240	0	1	0	0	1	0	0	0	0	0	0	0.2
	DOC_USP7_UBL2_3	ELME000394	1	2	0	0	0	0	0	0	1	3	0	0.4
	DOC_WW_Pin1_4	ELME000136	3	0	1	0	6	2	2	2	3	3	1	0.8
Ligand	LIG_14-3-3_CanoR_1	ELME000417	2	4	1	1	2	2	1	2	3	1	1	1.0
	LIG_14-3-3_CterR_2	ELME000418	1	0	0	0	0	0	0	0	0	0	0	0.1
	LIG_Actin_WH2_2	ELME000313	0	0	1	1	0	0	1	1	0	1	1	0.5
	LIG_APCC_ABBA_1	ELME000435	0	0	0	1	0	1	0	0	0	0	0	0.2
	LIG_BIR_II_1	ELME000285	1	1	1	1	1	1	1	1	1	1	1	1.0
	LIG_BIR_III_4	ELME000293	0	0	0	0	0	0	1	1	0	0	0	0.2
	LIG_BRCT_BRCA1_1	ELME000197	1	2	0	1	0	0	1	1	0	0	2	0.5
	LIG_BRCT_BRCA1_2	ELME000198	1	1	0	0	0	0	0	0	0	0	0	0.2
	LIG_CSL_BTD_1	ELME000410	1	0	1	1	1	1	0	0	0	0	0	0.5
	LIG_deltaCOP1_diTrp_1	ELME000459	0	0	0	0	0	0	0	0	1	0	0	0.1
	LIG_EH1_1	ELME000148	0	0	1	0	0	0	1	1	0	0	0	0.3
	LIG_eIF4E_1	ELME000317	0	0	0	0	1	0	0	0	0	0	1	0.2
	LIG_FHA_1	ELME000052	5	5	5	3	2	0	0	1	1	1	4	0.8
	LIG_FHA_2	ELME000220	1	1	0	1	3	2	0	0	0	1	0	0.5
	LIG_LIR_Apic_2	ELME000369	0	3	3	2	1	2	1	1	1	0	1	0.8
	LIG_LIR_Gen_1	ELME000368	2	1	1	1	2	1	0	1	1	1	0	0.8
	LIG_LIR_Nem_3	ELME000370	6	5	7	6	7	3	4	4	1	3	6	1.0
	LIG_LYPXL_S_1	ELME000294	0	0	0	0	0	0	0	0	0	0	1	0.1
	LIG_PCNA_PIPBox_1	ELME000140	0	0	1	0	0	0	0	0	0	0	0	0.1
	LIG_PCNA_yPIPBox_3	ELME000482	0	0	0	2	2	2	0	0	0	0	0	0.3
	LIG_Pex14_1	ELME000080	1	0	0	0	1	0	0	0	2	2	0	0.4
	LIG_Pex14_2	ELME000328	2	3	1	2	0	2	1	1	1	2	1	0.9
	LIG_PTB_Apo_2	ELME000122	0	0	0	2	1	0	1	1	1	1	0	0.5
	LIG_PTB_Phospho_1	ELME000095	0	0	0	1	1	0	1	1	0	0	0	0.4
	LIG_REV1ctd_RIR_1	ELME000450	0	0	0	0	0	0	0	0	1	0	0	0.1
	LIG_RPA_C_Fungi	ELME000382	0	0	0	0	0	0	1	1	0	1	0	0.3
	LIG_SH2_CRK	ELME000458	0	1	0	1	2	2	0	0	1	1	0	0.5
	LIG_SH2_GRB2like	ELME000084	0	0	0	0	0	0	0	0	0	0	1	0.1
	LIG_SH2_NCK_1	ELME000474	0	1	0	1	0	1	0	0	1	1	0	0.5
	LIG_SH2_PTP2	ELME000083	1	0	1	1	1	0	1	1	1	0	1	0.7
	LIG_SH2_SRC	ELME000081	1	0	2	1	1	1	1	1	1	0	2	0.8
	LIG_SH2_STAP1	ELME000465	2	1	0	0	1	1	0	0	1	1	1	0.6
	LIG_SH2_STAT3	ELME000163	2	2	1	0	1	1	1	1	0	0	1	0.7
	LIG_SH2_STAT5	ELME000182	3	1	3	2	3	2	3	3	3	2	5	1.0
	LIG_SH3_3	ELME000155	1	2	1	1	3	2	1	1	5	4	2	1.0
	LIG_SUMO_SIM_par_1	ELME000333	0	1	0	1	0	0	0	0	1	2	0	0.4
	LIG_SxIP_EBH_1	ELME000254	1	0	1	2	0	1	0	2	0	1	1	0.6
	LIG_TRAF2_1	ELME000117	1	1	0	1	1	2	1	1	0	0	0	0.6
	LIG_TRAF6	ELME000133	0	0	0	0	0	1	0	0	0	0	0	0.1
	LIG_TRFH_1	ELME000249	0	0	0	0	0	0	0	0	0	0	1	0.1
	LIG_TYR_ITIM	ELME000020	1	0	2	1	1	0	1	1	1	0	1	0.7
	LIG_UBA3_1	ELME000395	0	0	1	0	0	0	0	0	0	0	1	0.2
	LIG_Vh1_VBS_1	ELME000438	0	0	0	1	0	0	1	1	0	1	0	0.4
	LIG_WD40_WDR5_VDV_1	ELME000364	0	0	0	0	0	0	0	0	0	1	0	0.1
	LIG_WD40_WDR5_VDV_2	ELME000365	2	10	6	5	6	8	8	8	10	10	12	1.0
	LIG_WW_3	ELME000135	0	0	0	1	0	0	0	0	0	0	0	0.1
Modification	MOD_CDK_SPK_2	ELME000429	0	0	1	0	1	0	0	0	0	0	1	0.3
	MOD_CDK_SPxxK_3	ELME000428	0	0	0	0	1	1	0	0	1	1	0	0.4
	MOD_CK1_1	ELME000063	1	0	1	2	3	1	3	3	4	3	3	0.9
	MOD_CK2_1	ELME000064	3	2	1	4	3	2	0	0	0	1	0	0.6
	MOD_CMANNOS	ELME000160	0	0	0	0	1	0	0	0	0	0	0	0.1
	MOD_Cter_Amidation	ELME000093	1	1	0	0	0	0	0	0	0	0	0	0.2
	MOD_GlcNHglycan	ELME000085	1	3	3	3	2	0	2	2	4	2	5	0.9
	MOD_GSK3_1	ELME000053	3	1	3	4	2	2	1	1	0	4	6	0.9
	MOD_NEK2_1	ELME000336	2	3	3	2	2	1	4	4	1	2	3	1.0
	MOD_NEK2_2	ELME000337	0	0	1	1	1	1	0	0	1	0	1	0.5
	MOD_N-GLC_1	ELME000070	3	0	0	2	1	1	0	0	2	1	1	0.6
	MOD_N-GLC_2	ELME000079	0	0	1	0	2	0	0	0	0	0	1	0.3
	MOD_PIKK_1	ELME000202	5	3	2	1	2	0	0	0	2	1	0	0.6
	MOD_PK_1	ELME000065	0	2	0	0	0	1	0	0	0	0	1	0.3
	MOD_PKA_1	ELME000008	0	1	0	1	0	1	0	0	0	1	0	0.4
	MOD_PKA_2	ELME000062	1	1	1	1	1	2	0	2	1	0	2	0.8
	MOD_Plk_1	ELME000442	4	2	1	2	2	4	1	1	2	3	2	1.0
	MOD_Plk_4	ELME000444	1	2	3	2	2	2	0	0	1	2	0	0.7
	MOD_ProDKin_1	ELME000159	3	3	1	0	6	2	2	2	3	3	1	0.9
	MOD_SUMO_for_1	ELME000002	0	0	0	0	0	0	0	0	2	0	0	0.1
	MOD_SUMO_rev_2	ELME000393	1	1	0	0	0	0	0	0	0	0	0	0.2
Targeting	TRG_ENDOCYTIC_2	ELME000120	2	1	3	2	2	2	1	1	2	1	2	1.0
	TRG_LysEnd_APsAcLL_1	ELME000149	0	0	0	0	0	0	0	0	1	0	1	0.2
	TRG_NLS_MonoExtC_3	ELME000278	0	0	0	0	0	1	0	0	0	0	0	0.1
	TRG_Pf-PMV_PEXEL_1	ELME000462	1	1	1	1	1	1	1	1	1	1	1	1.0

GenBank accession numbers for endogenous integrase sequences are as follows: Endogenous retrovirus-K (ERVK in Homo sapiens; P10266.2), ERVK-8 pol protein-like (Macaca fascicularis; XP_015309771.1), Pol protein (Chlorocebus sabaeus; KFO35018.1), Pol protein (Fukomys darmarensis; BBC20786.1), Putative protein (Ochonta princeps; XP_012786727.1), ERVK-8 pol protein-like (Equus asinus; XP_014715024.1), ERVK-18 pol protein-like (Capra hircus; XP_017905435.1), Pol protein (Ovis aries; ABV71120.1), Putative protein (Zonotrichia albicollis; TRZ15504.1), Putative protein (Zosterops borbonicus; XP_014125095.1), Integrase (Onchocerca flexuosa; OZC05619.1).

. ELMs unique to ERVK IN, as well as ELM sites exhibiting motif consensus above 70% with other integrases, were annotated in Figure 1 and Figure 2.

2.3. STRING Analysis and KEGG Pathways

To identify potential ERVK IN binding partners based on ELM motifs, the names of interacting proteins were curated from each ELM reference page. When only a general interaction domain for a given ELM was listed, it was further linked to the InterPro database to curate a list of human proteins containing the interaction domain. Based on the 48 ELMs identified in ERVK IN, a total of 213 putative human protein interaction partners were identified (

Table A5. List of query proteins for STRING analysis based on ELM interaction motifs in ERVK integrase.

Category	ELM	STRING Predicted Interactor (Gene Name)	Network
Cleavage	CLV_C14_Caspase3-7	CASP3	•
		CASP7	•
	CLV_PCSK_KEX2_1 CLV_PCSK_PC1ET2_1 CLV_PCSK_SKI1_1	PCSK1	•
		PCSK2	•
		PCSK3	•
		PCSK4	•
		PCSK5	•
		PCSK6	•
		PCSK7	•
		PCSK8	•
		PCSK9	•
Docking site	DOC_CYCLIN_RXL_1	CCNA1	•
		CCNA2	•
		CCNB1	•
		CCNB2	•
		CCNB3	•
		CCNC	•
		CCND1	•
		CCND2	•
		CCND3	•
		CCNE1	•
		CCNE2	•
		CCNF	•
		CCNG1	•
		CCNG2	•
		CCNH	•
		CCNI	•
		CCNI2	•
		CCNJ	•
		CCNJL	•
		CCNK	•
		CCNL1	•
		CCNL2	•
		CCNO	•
		CCNP	•
		CCNT1	•
		CCNT2	•
		CCNY	•
		CCNYL1	•
		CNTD1	•
	DOC_MAPK_MEF2A_6	MAPK1	•
		MAPK3	•
		MAPK7	•
		MAPK11	•
		MAPK14	•
	DOC_PP1_RVXF_1	PPP1CA	•
		PPP1CB	•
		PPP1CC	•
		PPP3CA	•
		PPP3CB	•
		PPP3CC	•
	DOC_PP2B_LxvP_1	PPP3R1	•
	DOC_USP7_MATH_1 DOC_USP7_UBL2_3	USP7	•
	DOC_WW_Pin1_4	PCIF1	•
		PIN1	•
		SENP6	•
Ligand	LIG_14-3-3_CanoR_1 LIG_14-3-3_CterR_2	SFN	•
		YWHAB	•
		YWHAE	•
		YWHAG	•
		YWHAH	•
		YWHAQ	•
		YWHAZ	•
	LIG_BIR_II_1	BIRC2	•
		BIRC3	•
		BIRC5	•
		BIRC6	•
		BIRC7	•
		NAIP	•
		XIAP	•
	LIG_BRCT_BRCA1_1	BARD1	•
		BRCA1	•
		CTDP1	•
		DNTT	•
		ECT2	•
		LIG4	•
		MCPH1	•
		MDC1	•
		NBN	•
		PARP1	•
		PARP4	•
		PAXIP1	•
		PES1	•
		RBPJ	•
		REV1	•
		RFC1	•
		TOPBP1	•
		TP53BP1	•
		XRCC1	•
	LIG_FHA_1 LIG_FHA_2	AGGF1	•
		APLF	•
		APTX	•
		CEP170	•
		CEP170B	•
		CHEK2	•
		CHFR	•
		FHAD1	•
		FOXK1	•
		FOXK2	•
		KIF13A	•
		KIF13B
		KIF14	•
		KIF16B
		KIF1A	•
		KIF1B	•
		KIF1C
		MCRS1	•
		MDC1	•
		MKI67	•
		MLLT4	•
		NBN	•
		PHF12	•
		PPP1R8	•
		RNF8	•
		SLC4A1AP	•
		SLMAP	•
		SNIP1	•
		STARD9	•
		TCF19	•
		TIFA	•
TIFAB		•
LIG_CSL_BTD_1	CHEK2	•
	MRC1	•
	RAD9A	•
	XRCC1	•
	XRCC4	•
LIG_LIR_Gen_1 LIG_LIR_Nem_4	GABARAP
	GABARAPL1
	GABARAPL2	•
	MAP1LC3A
	MAP1LC3B
	MAP1LC3B2	•
	MAP1LC3C
LIG_Pex14_1	PEX13	•
	PEX14	•
LIG_SH2_PTP2	PLCG1	•
	PTPN11	•
LIG_SH2_SRC	BLK	•
	FGR	•
	FRK	•
	FYN	•
	HCK	•
	LCK	•
	LYN	•
	SRC	•
	YES1	•
LIG_SH2_STAP1	STAP1	•
LIG_TYR_ITIM	ABL1	•
	ABL2	•
	FYN	•
	LCK	•
	MATK	•
	PI3KCA	•
	PLCG1	•
	SH2D1A	•
	SHF	•
	PTPN6	•
	PTPN11	•
	SRC	•
	SYK	•
LIG_SH2_STAT3	STAT3	•
LIG_SH2_STAT5	STAT5A	•
	STAT5B	•
LIG_SH3_3	ARHGEF7	•
	CTTN	•
LIG_SxIP_EBH_1	MAPRE1	•
	MAPRE2	•
	MAPRE3	•
LIG_TRAF2_1	TRAF2	•
LIG_WD40_WDR5_VDV_2	WDR5	•
Modification	MOD_CK1_1	CSNK1A1	•
	MOD_CK2_1	CSNK2A1	•
	MOD_GSK3_1	GSK3A	•
		GSK3B	•
	MOD_N-GLC_1	DDOST	•
	MOD_NEK2_1	NEK2	•
	MOD_PIKK_1	ATM	•
		ATR	•
		mTOR	•
		PRKDC	•
		SMG1	•
		TRRAP	•
	MOD_PKA_2	PAK1	•
		PRKACA	•
		PRKACB	•
		PRKACG	•
		PRKCA	•
		PRKCB	•
		PRKCE	•
		PRKCG	•
		PRKCH	•
		PRKCI	•
		PRKCQ	•
		PRKCZ	•
	MOD_Plk_1 MOD_Plk_4	PLK1	•
		PLK2	•
		PLK3	•
		PLK4	•
	MOD_ProDKin_1	MAPK11	•
		MAPK12
		MAPK13
		MAPK14	•
	MOD_SUMO_rev_2	SUMO2	•
Targeting	TRG_ENDOCYTIC_2	AP1M1	•
		AP2M1	•
		AP3M1	•
		AP3M2
		AP4M1	•
		AP5M1
		ARCN1
		FCHO1	•
		FCHO2	•
		SGIP1	•
		STON1	•
		STON2	•
	TRG_Pf-PMV_PEXEL_1	None

Circle symbol: denotes protein depicted in network analysis in Figure 3.

). The list was submitted to STRING (String Consortium; version 11.0; Software for Technical Computation; 2020, https://string-db.org/, accessed on 16 May 2021) for network analysis. Full network analysis was performed using Experiment and Databases as active interaction sources. Nodes indicate submitted query proteins only, with edges indicating confidence lines with a minimum interaction score of 0.9 (highest confidence). Query proteins unlinked to the network were excluded from analysis. A payload list was used to color hub proteins based on cellular function. KEGG pathways associated with the network analysis (E value < 1.0 × 10⁻⁵) were presented in a heatmap using GraphPad Prism (version 9.1.1) software, and the full list of KEGG pathways is presented in

Table A6. Full list of KEGG pathways identified in the STRING analysis of the ERVK integrase interactome.

KEGG Term ID	Term Description	Observed Gene Count	Background Gene Count	Strength	False Discovery Rate
hsa04218	Cellular senescence	28	156	1.27	2.30 × 10−23
hsa05203	Viral carcinogenesis	29	183	1.21	3.47 × 10−23
hsa04110	Cell cycle	25	123	1.32	2.38 × 10−22
hsa04114	Oocyte meiosis	23	116	1.31	2.13 × 10−20
hsa05169	Epstein–Barr virus infection	24	194	1.11	4.01 × 10−17
hsa05205	Proteoglycans in cancer	23	195	1.08	4.79 × 10−16
hsa04650	Natural killer cell-mediated cytotoxicity	19	124	1.2	3.96 × 10−15
hsa04068	FoxO signaling pathway	19	130	1.18	7.58 × 10−15
hsa04750	Inflammatory mediator regulation of TRP channels	17	92	1.28	8.12 × 10−15
hsa05167	Kaposi’s sarcoma-associated herpesvirus infection	21	183	1.07	1.30 × 10−14
hsa04720	Long-term potentiation	15	64	1.38	1.77 × 10−14
hsa05161	Hepatitis B	19	142	1.14	2.19 × 10−14
hsa05206	MicroRNAs in cancer	19	149	1.12	4.49 × 10−14
hsa04370	VEGF signaling pathway	14	59	1.39	1.11 × 10−13
hsa04660	T cell receptor signaling pathway	16	99	1.22	2.39 × 10−13
hsa04012	ErbB signaling pathway	15	83	1.27	3.37 × 10−13
hsa04611	Platelet activation	17	123	1.15	3.37 × 10−13
hsa04921	Oxytocin signaling pathway	18	149	1.09	4.19 × 10−13
hsa04115	p53 signaling pathway	14	68	1.33	4.53 × 10−13
hsa05200	Pathways in cancer	29	515	0.76	6.20 × 10−13
hsa05166	HTLV-I infection	21	250	0.94	1.82 × 10−12
hsa04933	AGE-RAGE signaling pathway in diabetic complications	15	98	1.2	2.24 × 10−12
hsa04510	Focal adhesion	19	197	1	2.57 × 10−12
hsa04659	Th17 cell differentiation	15	102	1.18	3.46 × 10−12
hsa05031	Amphetamine addiction	13	65	1.31	3.95 × 10−12
hsa04728	Dopaminergic synapse	16	128	1.11	4.97 × 10−12
hsa04658	Th1 and Th2 cell differentiation	14	88	1.21	7.29 × 10−12
hsa05165	Human papillomavirus infection	22	317	0.85	1.27 × 10−11
hsa04914	Progesterone-mediated oocyte maturation	14	94	1.19	1.52 × 10−11
hsa04310	Wnt signaling pathway	16	143	1.06	2.02 × 10−11
hsa04390	Hippo signaling pathway	16	152	1.04	4.56 × 10−11
hsa04062	Chemokine signaling pathway	17	181	0.99	5.10 × 10−11
hsa04917	Prolactin signaling pathway	12	69	1.25	1.03 × 10−10
hsa04662	B cell receptor signaling pathway	12	71	1.24	1.35 × 10−10
hsa04919	Thyroid hormone signaling pathway	14	115	1.1	1.47 × 10−10
hsa04064	NF-kappa B signaling pathway	13	93	1.16	1.57 × 10−10
hsa04270	Vascular smooth muscle contraction	14	119	1.08	2.11 × 10−10
hsa04360	Axon guidance	16	173	0.98	2.24 × 10−10
hsa05162	Measles	14	133	1.04	7.76 × 10−10
hsa05223	Non-small cell lung cancer	11	66	1.23	9.08 × 10−10
hsa04666	Fc gamma R-mediated phagocytosis	12	89	1.14	1.18 × 10−9
hsa04380	Osteoclast differentiation	13	124	1.03	3.48 × 10−9
hsa01521	EGFR tyrosine kinase inhibitor resistance	11	78	1.16	4.18 × 10−9
hsa04010	MAPK signaling pathway	18	293	0.8	5.87 × 10−9
hsa04931	Insulin resistance	12	107	1.06	7.37 × 10−9
hsa04910	Insulin signaling pathway	13	134	1	7.61 × 10−9
hsa04724	Glutamatergic synapse	12	112	1.04	1.14 × 10−8
hsa04151	PI3K-Akt signaling pathway	19	348	0.75	1.17 × 10−8
hsa04912	GnRH signaling pathway	11	88	1.11	1.17 × 10−8
hsa04664	Fc epsilon RI signaling pathway	10	67	1.19	1.30 × 10−8
hsa04071	Sphingolipid signaling pathway	12	116	1.03	1.51 × 10−8
hsa04722	Neurotrophin signaling pathway	12	116	1.03	1.51 × 10−8
hsa01522	Endocrine resistance	11	95	1.08	2.24 × 10−8
hsa04014	Ras signaling pathway	15	228	0.83	5.08 × 10−8
hsa04210	Apoptosis	12	135	0.96	6.74 × 10−8
hsa05152	Tuberculosis	13	172	0.89	1.01 × 10−7
hsa04540	Gap junction	10	87	1.07	1.11 × 10−7
hsa05221	Acute myeloid leukemia	9	66	1.15	1.42 × 10−7
hsa05214	Glioma	9	68	1.13	1.74 × 10−7
hsa05222	Small cell lung cancer	10	92	1.05	1.74 × 10−7
hsa01524	Platinum drug resistance	9	70	1.12	2.13 × 10−7
hsa04215	Apoptosis—multiple species	7	31	1.37	2.13 × 10−7
hsa04926	Relaxin signaling pathway	11	130	0.94	3.72 × 10−7
hsa04015	Rap1 signaling pathway	13	203	0.82	5.46 × 10−7
hsa04960	Aldosterone-regulated sodium reabsorption	7	37	1.29	5.77 × 10−7
hsa04668	TNF signaling pathway	10	108	0.98	6.23 × 10−7
hsa04261	Adrenergic signaling in cardiomyocytes	11	139	0.91	6.58 × 10−7
hsa05145	Toxoplasmosis	10	109	0.98	6.58 × 10−7
hsa04725	Cholinergic synapse	10	111	0.97	7.54 × 10−7
hsa05120	Epithelial cell signaling in Helicobacter pylori infection	8	66	1.1	1.53 × 10−6
hsa04340	Hedgehog signaling pathway	7	46	1.2	1.97 × 10−6
hsa04961	Endocrine and other factor-regulated calcium reabsorption	7	47	1.19	2.22 × 10−6
hsa04066	HIF-1 signaling pathway	9	98	0.98	2.44 × 10−6
hsa04520	Adherens junction	8	71	1.06	2.44 × 10−6
hsa04916	Melanogenesis	9	98	0.98	2.44 × 10−6
hsa05225	Hepatocellular carcinoma	11	163	0.84	2.56 × 10−6
hsa04621	NOD-like receptor signaling pathway	11	166	0.83	2.99 × 10−6
hsa05014	Amyotrophic lateral sclerosis (ALS)	7	50	1.16	2.99 × 10−6
hsa05220	Chronic myeloid leukemia	8	76	1.04	3.62 × 10−6
hsa05020	Prion diseases	6	33	1.27	4.44 × 10−6
hsa04020	Calcium signaling pathway	11	179	0.8	5.69 × 10−6
hsa04670	Leukocyte transendothelial migration	9	112	0.92	6.16 × 10−6
hsa04726	Serotonergic synapse	9	112	0.92	6.16 × 10−6
hsa04723	Retrograde endocannabinoid signaling	10	148	0.84	7.20 × 10−6
hsa04727	GABAergic synapse	8	88	0.97	9.28 × 10−6
hsa04934	Cushing’s syndrome	10	153	0.83	9.30 × 10−6
hsa04924	Renin secretion	7	63	1.06	1.09 × 10−5
hsa04024	cAMP signaling pathway	11	195	0.76	1.14 × 10−5
hsa04657	IL-17 signaling pathway	8	92	0.95	1.20 × 10−5
hsa04022	cGMP-PKG signaling pathway	10	160	0.81	1.28 × 10−5
hsa04140	Autophagy—animal	9	125	0.87	1.28 × 10−5
hsa04630	Jak-STAT signaling pathway	10	160	0.81	1.28 × 10−5
hsa04713	Circadian entrainment	8	93	0.95	1.28 × 10−5
hsa05146	Amoebiasis	8	94	0.94	1.32 × 10−5
hsa05215	Prostate cancer	8	97	0.93	1.62 × 10−5
hsa05231	Choline metabolism in cancer	8	98	0.92	1.72 × 10−5
hsa04371	Apelin signaling pathway	9	133	0.84	1.94 × 10−5
hsa04930	Type II diabetes mellitus	6	46	1.13	2.04 × 10−5
hsa03450	Non-homologous end-joining	4	13	1.5	3.50 × 10−5
hsa04072	Phospholipase D signaling pathway	9	145	0.81	3.61 × 10−5
hsa05226	Gastric cancer	9	147	0.8	3.96 × 10−5
hsa05210	Colorectal cancer	7	85	0.93	5.70 × 10−5
hsa04217	Necroptosis	9	155	0.78	5.77 × 10−5
hsa04730	Long-term depression	6	60	1.01	7.67 × 10−5
hsa04925	Aldosterone synthesis and secretion	7	93	0.89	9.48 × 10−5
hsa04530	Tight junction	9	167	0.74	9.70 × 10−5
hsa05131	Shigellosis	6	63	0.99	9.70 × 10−5
hsa05010	Alzheimer’s disease	9	168	0.74	9.98 × 10−5
hsa05164	Influenza A	9	168	0.74	9.98 × 10−5
hsa05160	Hepatitis C	8	131	0.8	0.00011
hsa03440	Homologous recombination	5	40	1.11	0.00012
hsa04922	Glucagon signaling pathway	7	100	0.86	0.00014
hsa05140	Leishmaniasis	6	70	0.95	0.00016
hsa05168	Herpes simplex infection	9	181	0.71	0.00016
hsa04971	Gastric acid secretion	6	72	0.93	0.00018
hsa04918	Thyroid hormone synthesis	6	73	0.93	0.00019
hsa05133	Pertussis	6	74	0.92	0.0002
hsa05212	Pancreatic cancer	6	74	0.92	0.0002
hsa05224	Breast cancer	8	147	0.75	0.00021
hsa04150	mTOR signaling pathway	8	148	0.75	0.00022
hsa05110	Vibrio cholerae infection	5	48	1.03	0.00025
hsa04810	Regulation of actin cytoskeleton	9	205	0.66	0.00037
hsa04911	Insulin secretion	6	84	0.87	0.00037
hsa05130	Pathogenic Escherichia coli infection	5	53	0.99	0.00037
hsa04970	Salivary secretion	6	86	0.86	0.00041
hsa05416	Viral myocarditis	5	56	0.96	0.00046
hsa05032	Morphine addiction	6	91	0.83	0.00053
hsa05213	Endometrial cancer	5	58	0.95	0.00053
hsa04915	Estrogen signaling pathway	7	133	0.73	0.00063
hsa05418	Fluid shear stress and atherosclerosis	7	133	0.73	0.00063
hsa04213	Longevity regulating pathway—multiple species	5	61	0.93	0.00064
hsa04550	Signaling pathways regulating pluripotency of stem cells	7	138	0.72	0.00076
hsa05211	Renal cell carcinoma	5	68	0.88	0.001
hsa04920	Adipocytokine signaling pathway	5	69	0.87	0.0011
hsa05219	Bladder cancer	4	41	1	0.0013
hsa04211	Longevity regulating pathway	5	88	0.77	0.0029
hsa04923	Regulation of lipolysis in adipocytes	4	53	0.89	0.0031
hsa04120	Ubiquitin mediated proteolysis	6	134	0.66	0.0033
hsa04070	Phosphatidylinositol signaling system	5	97	0.72	0.0043
hsa05034	Alcoholism	6	142	0.64	0.0043
hsa05142	Chagas disease (American trypanosomiasis)	5	101	0.71	0.005
hsa04620	Toll-like receptor signaling pathway	5	102	0.7	0.0051
hsa04932	Non-alcoholic fatty liver disease (NAFLD)	6	149	0.62	0.0053
hsa05230	Central carbon metabolism in cancer	4	65	0.8	0.0059
hsa03410	Base excision repair	3	33	0.97	0.0066
hsa05143	African trypanosomiasis	3	34	0.96	0.0071
hsa04622	RIG-I-like receptor signaling pathway	4	70	0.77	0.0075
hsa05218	Melanoma	4	72	0.76	0.0081
hsa05216	Thyroid cancer	3	37	0.92	0.0087
hsa05202	Transcriptional misregulation in cancer	6	169	0.56	0.0091
hsa04152	AMPK signaling pathway	5	120	0.63	0.0093
hsa04962	Vasopressin-regulated water reabsorption	3	44	0.85	0.0132
hsa05132	Salmonella infection	4	84	0.69	0.0132
hsa03015	mRNA surveillance pathway	4	89	0.67	0.0157
hsa04913	Ovarian steroidogenesis	3	49	0.8	0.0171
hsa05030	Cocaine addiction	3	49	0.8	0.0171
hsa03460	Fanconi anemia pathway	3	51	0.78	0.0187
hsa05134	Legionellosis	3	54	0.76	0.0215
hsa04137	Mitophagy—animal	3	63	0.69	0.0314
hsa04714	Thermogenesis	6	228	0.43	0.0314
hsa04927	Cortisol synthesis and secretion	3	63	0.69	0.0314
hsa04976	Bile secretion	3	71	0.64	0.0413
hsa04136	Autophagy—other	2	30	0.84	0.045

.

3. Results

3.1. Characterization of Eukaryotic Linear Motifs in ERVK Integrase and Other Betaretroviral Integrases

To establish which exogenous and endogenous retroviruses contain integrase sequences most similar to ERVK IN, we performed BLASTp searches using the nr, mo, and tsa NCIB databases. As expected, exogenous Betaretroviruses were identified through BLASPp search, which included multiple hits for Mouse mammary tumor virus (MMTV), Mason–Pfizer monkey virus (M-PMV), Enzootic Nasal Tumor Virus (ENTV), and Jaagsiekte sheep retrovirus (JSRV) (Table A1).

Eukaryotic linear motif (ELM) analysis of a representative sequence from each genus was compared with ERVK IN and revealed the conservation of select protein motifs (Table 1, Figure 1). Apart from the conservation of the HHCC region and DDE active site motif, all betaretroviral integrases also contained many interaction motifs related to DRR, including Pin1 via [ST]P WW domain interaction motifs [49], PP1c docking motif for target dephosphorylation [50], and a S-X-X-S/T CK1 phosphorylation site [51]. All INs except for MMTV contained a low-affinity BRCA1 carboxy-terminal BRCT domain binding motif (CSKAF, aa. 126–132). Betaretroviral INs were also predicted to be phosphorylated by the cell cycle checkpoint kinases NEK2 [52] and PLK-1 [53], as well as interact with a canonical arginine-containing phospho-motif within cell cycle regulating 14-3-3 proteins [54]. Numerous cell signaling protein interactions were predicted including YXXQ motifs for the SH2 domain binding of STAT3 [55], additional SH2 binding motifs related to STAT5 [56] and SRC family kinases [57], SH3 binding motifs with non-canonical class I recognition specificity [58], an IAP-binding motif (aa. 1–4) for interaction with inhibitor of apoptosis proteins (IAP) [59], an ITIM motif [60], several GSK3 phosphorylation sites [61], and proline-directed ERK/p38 MAPK phosphorylation sites [62]. In addition, most betaretroviral IN enzymes contained features related to protein trafficking, such as a Wxxx[FY] motif (aa. 133–137 in ERVK, MMTV, ENTV, and JRSV) that binds Pex13 and Pex14 for peroxisomal import [63], a SxIP motif (aa. 137–150) that binds to EBH domains in end-binding proteins involved in microtubule transport [64], and a tyrosine-based YXXØ sorting signal (aa. 75–78) for interaction with the μ-subunit of adaptor protein complex [65] and a PEXEL-like motif [66]. The DDE region displayed the most consistent pattern of conserved ELMs among the betaretroviral INs. It is important to note that despite the similar complement of ELM motifs in betaretroviral integrases, many were positioned at sites differently than in ERVK IN. Additional ELMs and their motif frequencies in individual betaretroviral integrases are listed in Table 1.

3.2. Characterization of Eukaryotic Linear Motifs in ERVK Integrase and Other Endogenous Integrases

ERVK integrase-like sequences were found in boreoeutherians, including the Euarchontoglires (primates, rodents, and pikas), and Laurasiatherians (ungulates), along with other clades including Euteleostomi (birds) and Protostomes (worms, insects, and water fleas) (Table A2, Table A3 and Table A4). Results ranged from 26.43 to 83.77% identity and E values ranged from 0.001 to 2.0 × 10⁻¹²⁷, demonstrating a high degree of similarity with ERVK IN.

The conservation of ELM motifs was apparent (Table 2, Figure 2), including DDR-related canonical 14-3-3 interaction motifs and WDR5 interaction, cell signaling associated with USP7 binding, IAP-binding motif, STAT5 binding motifs, SH3 protein interaction, as well as phosphorylation sites for CK proteins, GSK3, NEK2, polo-like kinases, and p38. Many LIR motifs for engaging Atg8 proteins during selective autophagy were also apparent. Finally, all IN displayed Pex14 binding motifs and potential to interact with the μ-subunit of the adaptor protein complex. Additional ELMs and their motif frequencies in individual endogenous ERVK-like INs are listed in Table 2.

ELMs within endogenous IN but not or rarely identified in ERVK IN were also noted. An Apicomplexa-specific variant of the canonical LIR motif that binds to Atg8 protein family members was present in all endogenous INs except for ERVK IN. In addition, WDR5 binding motifs were much more prevalent in endogenous INs (5–12 sites) other than ERVK IN (only two sites). ERVK IN contained a single MAPK docking site for ERK/p38, whereas other endogenous INs contained several other motifs for MAPK interaction. Lastly, only human and macaque ERVK INs displayed high-affinity BRCT domain interaction motifs.

3.3. Unlike Similar Enzymes, the ERVK Integrase Contains Distinct ELM Signatures

Two motifs in ERVK IN stand out as unique to this virus, while other signatures are enriched in ERVK IN as compared with similar integrases.

3.3.1. ERVK Integrase has a High-Affinity BRCA1 Binding Site

Among all the integrases examined, only ERVK IN in human and macaque harbored a high-affinity binding site for the BRCT domain of BRCA1 (aa. 125–131, CSKAFQK) (Table 1 and Table 2, Figure 1 and Figure 2).

3.3.2. ERVK Integrase C-Terminus Contains a 14-3-3 Binding RASTE Motif

Although 14-3-3 protein binding was predicted as conserved among ERVK-like integrases, only ERVK IN contained a C-terminal RASTE motif (aa. 276–280) mediating strong interaction with 14-3-3 proteins (Table 1, Figure 1). This suggests a putative ERVK IN interaction with 14-3-3 proteins through both canonical phospho-sites and a C-terminal phospho-site.

3.3.3. ERVK Integrase Is Likely Post-Translationally Sumoylated

Unlike all other INs examined, only ERVK and MMTV contain a C-terminal inverted version (D/ExKphi) of the canonical sumoylation motif [67]. Considering that sumoylation often causes re-localization of nuclear proteins, this modification may be related to ERVK IN nuclear positioning, association with chromatin, and ultimately successful integration of viral DNA [68,69].

3.3.4. ERVK Integrase Exhibits Enhanced Interaction Potential with DDR Proteins

Phospho-Ser/Thr binding domain proteins are key hub proteins in cell cycle regulation and DDR, and they include 14-3-3 proteins, WW domains, Polo-box domains, WD40 repeats, BRCA1 carboxy-terminal (BRCT) domains, and Forkhead-associated (FHA) domains [54], all of which are interacting domains of ELMs identified in ERVK IN (Table 1 and Table 2, Figure 1). Additionally, ERVK IN contained five (ST)Q motifs, which are potential phosphorylation sites for PIKK proteins, such as DDR-related proteins ATR, ATM, DNA-PK, and multi-functional protein mTOR [70]. As compared with exogenous betaretroviruses and endogenous ERVK-like integrases, ERVK IN displayed a greater number of DDR-related motifs: FHA domain protein interaction sites (6), PLK-1 phosphorylation sites (4), and PP1c docking motif for target dephosphorylation (3) [50]. In contrast to MMTV, ENTV, JSRV, and most other endogenous integrases, fewer WD40 repeat domain WDR5 interaction sites were found in ERVK IN (2 vs. 5–12 sites each). This suggests ERVK IN has potentially shifted away from WDR5 interaction in favor of BRCA1 (or BRCT domain) interaction as a means to interact with the DDR pathway [54,71].

3.3.5. ERVK Integrase Contains Canonical Selective Autophagy Motifs

Unlike any of the exogenous betaretroviruses, only ERVK IN and some endogenous integrases contained canonical LIR motifs (ELME000368) for binding Atg8 protein family members (Table 1 and Table 2, Figure 2). All endogenous INs contained nematode-specific LIR motifs (ELME000370). Additionally, most endogenous INs housed Apicomplexa-specific LIR motifs (ELME000369), whereas ERVK IN did not.

3.4. ERVK Interactome Reveals Association with a Diversity of Cellular Pathways

Based on ELMs identified in ERVK IN, a curated list of potential interacting proteins was generated and used to build an ERVK IN interactome network using STRING software (Figure 3). The ERVK IN network contained 189 nodes and 692 edges (expected number of edges 222), resulting in a significant PPI enrichment (p < 1.0 × 10⁻¹⁶). Only direct interactor query proteins are shown without links to second shell interactions. To illustrate key nodes and hub proteins, select network proteins were colored based on function related to DDR, cell cycle, apoptosis, cell signaling, or cellular transport. A complete list of the KEGG pathways significantly associated with the network is presented in Table A6.

3.4.1. Many DNA Damage Response Proteins Are Potential ERVK Integrase Interactors

Gene ontology (GO) biological processes that were significantly enriched in the network included cellular response to DNA damage stimulus (p < 4.4 × 10⁻¹⁸), DNA repair (p < 4.24 × 10⁻¹²), DNA damage checkpoint (p < 4.89 × 10⁻¹²), double-strand break repair via non-homologous end joining (NHEJ) (p < 3.57 × 10⁻⁹), double-strand break repair (p < 1.06 × 10⁻⁸), and signal transduction in response to DNA damage (p < 2.12 × 10⁻⁸). Select BRCT domain containing proteins emerged as nodes with a higher-than-average degree of connections, including BRCA1, BARD1, NBN, MDC1, RCF1, TOPBP1, TP53BP1, and PAXIP1, while PARP1 and DRKDC (DNA-PK) appear to be hub proteins between DDR and apoptosis. The ERVK IN network also displayed four prominent DDR-related FHA proteins: CHEK2, NBN, MDC1, and RNF8.

3.4.2. ERVK Integrase Likely Modulates Cell Cycle Pathways

GO biological processes that were significantly enriched in the network included regulation of cell cycle (p < 1.45 × 10⁻³³), cell division (p < 1.12 × 10⁻²⁰), regulation of cyclin-dependent serine/threonine kinase activity (p < 6.86 × 10⁻²⁰), mitotic cell cycle (p < 2.78 × 10⁻¹⁷), regulation of apoptotic process (p < 1.45 × 10⁻¹⁷), and cell cycle checkpoint (p < 6.72 × 10⁻¹²). Many cyclins and 14-3-3 proteins were identified in the network and are listed in Table A5. IAP-containing protein BIRC5 (also known as survivin) was also identified, which is suggestive of negative regulation of programmed cell death pathways [72]. PLK1 and NEK2 were also tied into the cell cycle framework and are both regulators of mitosis, in addition to displaying oncogenic properties [73,74].

3.4.3. Cell Signaling Pathways Associated with the ERVK Interactome

Among the potential signaling pathways often targeted by retroviruses, ERVK IN-associated cascades emerged as Forkhead box O (FoxO) signaling [75], p53 signaling [76], ErbB signaling [77], Wnt signaling [78], modulation of kinase activity [79,80], and multiple aspects of immune signaling [81] (Figure 4). Within these pathways, prominent immune-related signaling intermediates included STAT3 [55], STAT5 [56], and TRAF2 [82]. The SH2 and SH3 containing tyrosine-protein kinase ABL1 (Abelson murine leukemia viral oncogene homolog 1 [57]) appears to be a key hub protein linking DDR and downstream signaling cascades.

3.4.4. ERVK Integrase May Utilize Specific Cellular Transport Systems

The ERVK interactome contains proteins related to cellular transport. EB1 (MAPRE1) is an end-binding (EB) protein connected with both cell cycle and signaling pathways and is functionally associated with the regulation of microtubule dynamics [83]. Adaptor protein complex 2 associated proteins (AP2M1 and CTTN) were identified and indicate a role in cargo internalization via clathrin-mediated endocytosis and actin dynamics [65,84]. Lastly, ERVK IN may interact with Pex14 and Pex13 independently of the main network for peroxisome import [63]. While these pathways were likely important for the ancestral exogenous ERVK to transverse the cell and mediate infection, it remains unclear how endogenous IN may interact with these systems.

3.5. Diseases and Pathways Implicated in the ERVK Integrase Interactome

3.5.1. Cancers

Viral carcinogenesis was the top KEGG pathway identified in the ERVK IN network analysis (strength 1.22, E value 3.7 × 10⁻²³), with 29 of 183 proteins represented (Figure 4). KEGG pathways for several known ERVK-associated cancers were also identified, including lung cancer [85], myeloid leukemia [86], and hepatocellular carcinoma [87] (Figure 4). Glioma was also identified, yet ERVK is downregulated in this condition [88]. Aligned with cellular transformation, proteins associated with cell cycle were also over-represented in the pathway analysis, which are specifically related to the cyclin docking site ELM (DOC_CYCLIN_RxL_1) and numerous FHA domain protein interaction sites (LIG_FHA_1 and LIG_FHA_2) in ERVK IN (Table 1 and Table 2, Figure 1 and Figure 2).

3.5.2. Neurological Disease

KEGG pathways for several ERVK-associated neurological conditions were identified, including ALS [9,12], Alzheimer’s disease [89], and prion disease [90] (Figure 4). Specifically, long-term potentiation and synaptic neurotransmitter release (dopaminergic, glutamatergic, cholinergic, serotonergic, and GABAergic) were associated with the ERVK IN interactome.

3.5.3. Diabetes

The role of ERVK in diabetes remains contentious [91,92,93,94]. However, network analysis suggests that the ERVK IN interactome is potentially linked to AGE-RAGE signaling in diabetic complications, insulin signaling, and insulin resistance (Figure 4).

4. Discussion

ERVK expression has been repeatedly associated with human disease states including cancer, neurological disease, and diabetes. By exploring the potential ERVK integrase interactome, we can postulate how this viral symbiont may contribute to disease pathogenesis via interaction with key proteins and pathways. Our analysis reveals that viral carcinogenesis and modulation of the DNA damage response are the most likely pathways to be pathologically associated with ERVK IN expression.

Retroviral integrase activity causes DNA lesions in the host genome as part of the proviral integration process [19]; therefore, interactions with DDR pathways are to be expected. Several DDR proteins have been shown to be essential for provirus suture into the host genome and maintenance of genome fidelity [19]. Yet, the impairment of select aspects of DDR has also been documented in exogenous retroviral infections, including HIV [95,96] and HTLV-1 [97,98]. This may be driven by the fact that NHEJ proteins also play an essential role in innate immune recognition of retroviral cytosolic ssDNA intermediates and dsDNA pre-integration complexes [98,99]. Thus, retroviruses must balance the benefits and drawbacks of DDR outcomes through the engagement and modulation of specific proteins.

BRCT domain, FHA domain, and 14-3-3 proteins work in concert during the DNA damage response (reviewed in [100]). Many of these DDR proteins are also cellular targets of retroviruses and oncogenic viruses [98,101,102,103,104]. BRCA1 BRCT domains recognize phosphopeptides based on a pSXXF motif, but XX residues and the surrounding amino acids also impact binding affinity and selectivity [105]. All the betaretroviral INs examined showed the capacity to interact with BRCT domains. However, only ERVK IN displayed a high affinity (S.F.K) BRCA1 BRCT domain binding site; the only other similar ELM structure is found in the DDR protein Fanconi anemia group J protein (FACJ/BACH1) [106]. It is also possible that dual anchoring onto the ERVK IN using both a BRCT domain and an FHA domain found in NBN or MDC1 may strengthen protein–protein interactions.

The utilization and evasion of 14-3-3 proteins are common among many viruses [104]. ERVK IN is unique in having a C-terminal RASTE motif, in addition to two other canonical arginine containing phospho-motifs recognized by 14-3-3 proteins. Given that an elevated expression of 14-3-3 proteins occurs in both cancers and neurodegenerative diseases [107,108], ERVK IN interaction with 14-3-3 protein members may be related to either modulation of the cell cycle and oncogenesis or regulation of protein aggregation, respectively. The deregulation of 14-3-3 and RAF kinase interaction can also lead to inappropriate downstream MAPK activity (associated with oncogenesis) [54,109] and may be an aspect to consider for the predicted ERVK IN network.

ABL1 appears to be a key hub protein linking DDR and downstream signaling cascades. Interestingly, DDR is known to be a rapid driver of ABL1 activation [110]. The ablation of ABL1 reduces retrovirus integration [111,112], while active ABL1 can turn on the HIV promoter independently of HIV Tat [113]. Putative interaction between ERVK IN and ABL1 may have been important for ERVK integration into germline cells, and it may additionally play a role in ERVK expression, specifically in neurodegenerative disease displaying enhanced ABL1 activity [114,115].

DDR is intimately tied to innate immune response, specifically NF-κB activation [116]. Considering ERVK’s dependence on NF-κB for driving its own expression [11], it is conceivable that ERVK IN plays a role in preparing the host cell for viral transcription. 14-3-3ϵ activity is key in driving ATM-TAK1-mediated NF-κB signaling during DDR [117,118]; thus, the predicted ERVK IN interaction with 14-3-3ϵ (YWHAE) may be a mechanism to favor viral transcription. The MAPK p38 was also predicted to both phosphorylate and bind ERVK IN. This association may be linked to p38′s regulation of inflammatory signaling, as well as its capacity to enhance the transcriptional activity of NF-κB p65 via modulation of the acetyltransferase activity of coactivator p300 [119]. Sustained NF-κB activity is linked to oncogenesis [116] and ties into the strongest ERVK IN-linked KEGG pathway: viral carcinogenesis. However, enhanced ERVK IN-associated NF-κB signaling may also fit with inflammatory and neurodegenerative conditions.

ERVK IN stability and protein turnover is likely linked to its cellular protein partnerships. In the case of HIV, binding select cellular proteins such as LEDGF/p75 and Ku70 prevents integrase proteosomal degradation [120,121]. Similarly, c-Jun N-terminal kinase (JNK) S⁵⁷ phosphorylation of the core domain can make HIV IN a target for Pin1, thus enhancing its stability and activity [122,123]. In this study, Pin1′s WW domain was predicted to be an interactor based on three [ST]P motifs in the C-terminal portion of ERVK IN. This raises the possibility that similar to many other viral proteins [124], ERVK IN may be stabilized through Pin1 interaction. The functional significance of this interaction may underlie how elevated levels of ERVK IN are maintained and potentially drive pathology in select diseases, such as ALS and cancer.

Distinct from other exogenous betaretroviruses, only ERVK IN and some endogenous integrases contained canonical LIR motifs for binding Atg8 protein family members. Mammalian Atg8-like proteins include LC3 and GABARAP families, which mediate selective autophagy, as well as play essential roles in antiviral defense and innate immune signaling [125]. However, it is often observed that viruses subvert autophagy processes to avoid viral protein clearance and repurpose Atg8 proteins as well as autophagosomal membranes for viral replication [125,126]. Considering the perturbances of autophagy in neurodegenerative disease [127,128], the interaction between ERVK IN and Atg8 proteins warrants further investigation.

Consistent with genomic instability profiles in cancer [129], ALS [130], and Alzheimer’s disease [131], the ERVK interactome analysis identified each of these conditions as significant KEGG pathways. Despite differences in clinical presentation, the molecular underpinnings in cancer and neurodegenerative disease are remarkably similar and include alterations in DDR [129,130,132], 14-3-3 expression [133,134], p53 signaling [135,136], p38 signaling [137,138], and Wnt signaling [139,140]—which are all KEGG pathways enriched in the ERVK IN network. AGE-RAGE signaling was also identified as a potential pathway associated with the ERVK IN interactome. Not only is this pathway implicated in diabetic complications [141], but it also plays a role in nuclear response to DNA damage [142], carcinogenesis [143], and inflammatory neurodegenerative diseases [144]. Collectively, our results point to ERVK IN driving a pattern of pathology that, depending on cellular context, may lead to carcinogenesis, neurodegeneration, or contribute to diabetic complications. However, the engagement of DDR can also have beneficial impacts on lifespan extension, depending on tissue context and host genotype; thus, non-pathogenic effects of ERVK IN should also be considered [145,146].

Apart from the importance of putative ERVK IN interaction partners, it is also important to consider which cellular proteins were not associated with the ERVK IN interactome. One interaction that was not predicted was with LEDGF/p75, and indeed, this interactor is limited to partnership with lentiviral integrases [147,148]. Another set of DDR proteins commonly found to impact retroviral integration and replication is the DNA-PK complex [99,149]. HIV integrase directly interacts with Ku70 [120]; while ERVK IN was predicted to be phosphorylated by DNA-PKcs (PRKDC), it contained no ELMs to suggest direct interaction with Ku80 or Ku70. Another apparent difference is the use of EB proteins in microtubule trafficking for HIV and ERVK. ERVK IN contained an SxIP motif that binds EBH domains, whereas HIV capsid conversely has EB-like motifs that interact with SxIP motifs in plus-end tracking protein (+TIP) [150]. These genus-specific distinctions are likely to emerge as important considerations for therapeutic targeting strategies and imply that pharmaceuticals geared toward HIV infection may not consistently translate for use in ERVK-associated disease.

Another consideration that stems from this study is the choice and caveats of using animal models in ERVK research. A diversity of animals outside of the primate lineage are host to ERVK IN-like sequences, such as rodents, ungulates, fish, and insects. Drosophila, a common model organism, also contained ERVK IN-like elements in their genome, specifically LTR retrotransposons flea and Xanthias, as identified by FlyBase (Table A4). The transposable element Xanthias is known to be active in D. melanogaster [151,152], and it shares a degree of similarity with ERVK IN. The presence and activity of these ERVs is an important factor to consider when performing experiments.

It is shocking how little we understand of the impact endogenous viral symbionts have on cellular functioning. Herein, we have predicted that ERVK IN may participate in the modulation of cellular pathways such as DDR, cell cycle regulation, and kinase signaling cascades by way of select protein interaction motifs. The main caveat of in silico predictions is the requirement for experimental validation; while research into ERVK IN is currently underway, this study suggests there remains a myriad of disease-related betaretroviral integrase interactions to explore.