1 infectious endogenous retroviruses in cats and emergence of recombinant viruses yukari

1

Infectious Endogenous Retroviruses in Cats and Emergence of Recombinant 1

Viruses 2

Yukari Anai1, Haruyo Ochi1, Shinya Watanabe1, So Nakagawa2,3, Maki Kawamura1, 3

Takashi Gojobori2 and Kazuo Nishigaki1,* 4

1Laboratory of Molecular Immunology and Infectious Disease, Joint Faculty of 5

Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, Japan 6

2Center for Information Biology and DNA Data Bank of Japan, National Institute of 7

Genetics, 1111 Yata, Mishima, Shizuoka, Japan 8

3present address: Department of Organismic and Evolutionary Biology, Harvard 9

University, Cambridge, MA 02138, USA; Research Fellow of the Japan Society for the 10

Promotion of Science 11

Running Title: Infectious ERV and emergence of recombinant virus 12

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*Corresponding author: Kazuo Nishigaki, DVM, Ph.D. 14

Joint Faculty of Veterinary Medicine, Yamaguchi University 15

1677-1 Yoshida, Yamaguchi, Japan 16

Phone: +81-83-933-5829 17

Fax: +81-83-933-5820 18

E-mail: [email protected] 19

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00280-12 JVI Accepts, published online ahead of print on 6 June 2012

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Abstract 23

Endogenous retroviruses (ERVs) comprise a significant percentage of the mammalian 24

genome, and it is poorly understood whether they will remain as inactive genomes or 25

emerge as infectious retroviruses. Although several types of ERVs are present in 26

domestic cats, infectious ERVs have not been demonstrated. Here we report a 27

previously uncharacterized class of endogenous gammaretroviruses, termed ERV-DCs, 28

that is present and hereditary in the domestic cat genome. We have characterized a 29

subset of ERV-DC proviral clones, numbered according to their genomic insertions. 30

One of these, ERV-DC10 located in the q12-q21 region on chromosome C1, is an 31

infectious gammaretrovirus capable of infecting a broad range of cells including human. 32

Our studies indicate that ERV-DC10 entered the genome of domestic cats in the recent 33

past and appeared to translocate to or reintegrate at a distinct locus as an infectious 34

ERV-DC18. Insertional polymorphism analysis revealed that 92 of 244 domestic cats 35

had ERV-DC10 on homozygous or heterozygous locus. ERV-DC-like sequences were 36

found in primate and rodent genomes, suggesting that these ERVs and recombinant 37

viruses such as RD-114 and BaEV may have originated from an ancestor of ERV-DC. 38

We also found that a novel recombinant virus, FeLV-D, was generated by ERV-DC env 39

transduction into feline leukemia virus in domestic cats. Our results indicate that 40

ERV-DCs behave as donors and/or acceptors in the generation of infectious, 41

recombinant viruses. The presence of such infectious endogenous retroviruses, which 42

could be harmful or beneficial to the host, may impact veterinary medicine and public 43

health. 44

45


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Introduction 46

Retroviruses are classified as exogenous or endogenous according to their mode of 47

transmission. Endogenous retroviruses (ERVs) are present in all vertebrate genomes and 48

are thought to be the remnants of ancestral germline infections by exogenous 49

retroviruses. ERVs make up a significant fraction of the mammalian genome (for 50

example, 8–10% of the human or mouse genomes) and are transmitted according to 51

Mendelian fashion (4,19,42). Most ERVs contain deleterious mutations – large deletions 52

and insertions of repetitive elements that render them unable to make infectious viral 53

particles, but these proviruses often are transcribed and produce functional gene 54

products (4). Domestic cats (Felis silvestris catus) are descended from Felis silvestris 55

lybica, the Middle Eastern wildcat (10), and are sometimes infected with feline 56

leukemia virus (FeLV), which is a gammaretrovirus that is horizontally transmitted 57

among domestic cats during grooming or through bites (13,14). The endogenous 58

counterparts of exogenous FeLV, termed enFeLVs, are integrated into the genomes of 59

the Felis genus. Primary colonization of the feline germline by FeLVs is believed to 60

have occurred in an ancestor of the domestic cat after separation from the leopard 61

lineage (2,13,31). Like most endogenous retroviruses, enFeLVs are non-infectious and 62

non-pathogenic (4). However, recombination occurs between inherited enFeLVs and 63

exogenous FeLVs (exFeLVs) following infection, which can give rise to new infectious 64

agents that may be pathogenic (13,29). In addition to enFeLV, other feline ERVs are 65

found in the domestic cat. The RD-114 ERV, which was originally isolated from human 66

rhabdomyosarcoma cells transplanted into fetal kittens (23), is a replication competent 67

retrovirus which shows a high level of homology to the baboon endogenous retrovirus 68

(BaEV); however, an infectious RD-114 provirus has not been demonstrated in the cat 69


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genome. MAC-1, which is a genetically transmitted type-C virus of primates, has 70

been reported as a feline non-infectious ERV (5,39); however, no further studies have 71

been reported. Another class of feline ERVs with high homology to the pol region of 72

BaEV and RD-114 has also been described and partially characterized as ECE1 and 73

FcEV (3,41). In the present study, we have further characterized this later class of 74

feline ERVs, which we term ERV-DCs. Our studies indicate that while most 75

ERV-DCs contain deletions and mutations that render them non-infectious, several 76

ERV-DCs are infectious and mobile in the domestic cat, using re-infection, translocation 77

to another locus or recombination with an exogenous retrovirus to amplify their progeny. 78

These infectious ERV-DCs can infect a wide range of mammalian cells, including 79

human. Most ERV-DCs are not yet fixed in the cat genome and have the potential to 80

generate recombinant retroviruses which could be transmitted among cats and other 81

species. 82

83

Materials & Methods 84

Samples. Blood for peripheral blood mononuclear cells (PBMC) isolation and spleen 85

and muscle tissues from domestic cats and Prionailurus bengalensis euptilurus were 86

collected at private veterinary hospitals (27) and Tsushima Wildlife Conservation Center 87

(TWCC) in Japan. 88

89

Expression vectors and cell lines. The env of FeLV-A/Glasgow-1 (pFGA5) (38), 90

FeLV-B/Gardner-Arnstein (pFGB)(25), FeLV-C/Sarma (pFSC)(33), FeLV-D/Ty26, 91


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FeLV-D/ON-T and ERV-DC10 were PCR-amplified with specific primers and cloned 92

into pFUΔss expression plasmid, which was a modified pFUSE-hIgG1-Fc2 vector 93

(Invitrogen, Carlsbad, CA). The nβ-gal gene (kindly provided by Masaaki Nakaya, 94

Yokohama City University, Kanagawa) was introduced into the pMXs-IP vector (18) 95

(kindly provided by Toshio Kitamura, The University of Tokyo, Tokyo) to produce 96

pMXs-nLIP. HEK293T cells were persistently infected with FeLV-A (clone33)(28), 97

FeLV-B (pFGB), FeLV-C (pFSC), ERV-DC10 (pDC10) or ERV-DC18(pDC18). 293T 98

cells expressing FeLV-D/ON-T were established by transfection with a full-length 99

FeLV-D/ON-T plasmid and then were infected with FeLV-A/clone33 for viral 100

interference assays. An env-negative packaging cell line containing a LacZ-coding 101

retroviral vector, designated GPLac, was also established. 102

103

RNA isolation and reverse transcription. Total RNA was isolated using a FastPure 104

RNA Kit and cDNA was synthesized with a PrimeScript® II 1st strand cDNA Synthesis 105

Kit (TaKaRa, Tokyo, Japan). Viral RNA was isolated from infected cell culture 106

supernatants using the QIAamp Viral RNA mini kit (QIAGEN, Tokyo, Japan). 107

108

PCR. Primers used in this study are listed in Supplementary Table 4. PrimeSTAR GXL 109

polymerase (TaKaRa), KOD FX Neo (TOYOBO, Japan) and PrimeSTAR HS DNA 110

polymerase (TaKaRa) were used for various cloning procedures. The latter two 111

enzymes were also used to screen for FeLV-D and monitor viral infection. KOD FX 112

Neo and GoTaq (Promega, Madison, WI) were used for genotyping insertional 113

polymorphisms. 114


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115

Southern blotting. A total of 7.5 µg sample DNA was isolated from peripheral blood 116

mononuclear cells (PBMCs) or tissue samples (34), digested with EcoRI and BamHI 117

and hybridized to a digoxigenin (DIG)-labelled FeLV-D/ON-T env probe generated by 118

PCR with primers Fe-9S and Fe-3R (Supplementary Table 4). A CSPD Luminescent 119

detection kit was used to visualize the bound probe (Roche Molecular Biochemicals, 120

Mannheim, Germany). 121

122

Cloning of ERV-DC proviruses. Splenic DNA from a single cat (ON-T) was digested 123

with EcoRI or Sau3AI and ligated to Lamda DASH II vectors (Stratagene, La Jolla, CA). 124

DNA libraries were screened with a DIG-labelled FeLV-D env probe. ERV-DC 125

proviruses and flanking sequences were obtained from the NCBI cat genome database; 126

other sequence data were obtained by screening genomic libraries. We PCR-amplified 127

full-length ERV-DC and partial ERV-DC proviruses from feline chromosomal DNA 128

using specific primers and cloned amplicons into pCR4 blunt-TOPO (Invitrogen, 129

Carlsbad, CA). Some flanking sequences of ERV-DCs were obtained by gene walking 130

using the Right Walk® kit (BEX CO., LTD., Tokyo, Japan). Pre-integration sites and 131

ERV-DC provirus were detected by PCR. 132

FeLV-D screening and cloning. DNA from FeLV-positive PBMCs or tumour tissues 133

was PCR-screened using primers Fe-14S and Fe-3R. Gene walking was performed to 134

obtain the flanking DNA sequences of FeLV-D/ON-T provirus using each Fe-14S (first 135

PCR) and Fe-2S (second PCR) primer with each adaptor primer (Right Walk kit, BEX 136


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CO., LTD.). After obtaining one sequence of the pre-integration sites, the full length 137

FeLV-D/ON-T proviral genome was PCR-amplified with Prime STAR HS (TaKaRa) 138

DNA polymerase and Fe-63S and Fe-73R primers and cloned into pCR4 blunt-TOPO. 139

Partial FeLV-D/Ty26, FeLV-D/ON-C, FeLV/44B and FeLV-D/re-T sequences were 140

obtained by PCR. 141

142

Immunoblotting. Immunoblotting was performed as previously described (40). 143

Primary antibodies were goat anti-RD114 (National Cancer Institute (NCI), Frederick, 144

MD), goat anti-FeLV gp70 (NCI) and mouse anti-beta actin (Santa Cruz Biotechnology, 145

Santa Cruz, CA). Secondary antibodies were horseradish peroxidase (HRP)-conjugated 146

anti-mouse IgG antibody (GE Healthcare Japan, Tokyo, Japan) or anti-goat IgG 147

antibody (Santa Cruz Biotechnology). 148

149

Infection assay. HEK293T cells were transfected with 4 μg ERV-DC10 or -DC18 150

plasmid DNA using Lipofectamine 2000 (Invitrogen). Viruses in the culture supernatant 151

collected 72 h post-transfection were detected by PCR. Supernatants (300 μl, 152

0.22-μm-filtered) from persistently infected HEK293T cells were used to infect Molt-4, 153

K562, HepG2, HeLa, MCF7, THP-1, Vero, Cos7, L929, BHK-21 and NIH3T3 cells 154

(3.65 × 105 cells/well) in the presence of polybrene (8 μg/ml). KwDM cells established 155

from dog mammary tumour were also used for infection assay. Cellular DNA was 156

collected and ERV-DC proviral DNA was detected by PCR using primers Fe-13S and 157

Fe-18R. 158


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GFP-containing pMXs-IP vector with or without ERV-DC10 or ERV-DC18 sequences 159

was also transfected into HEK293T cells. At 72 h post-transfection, CRFK, AH927 and 160

G355 cell lines were infected with the HEK293T supernatant in the presence of 161

polybrene (8 μg/ml). GFP-positive cells were determined by microscopy or FACS. 162

Viral preparation from feline chromosomal DNA. ERV-DC8, ERV-DC10, 163

ERV-DC14 or ERV-DC18 amplified by PCR with primers Fe-57S and Fe-54R for 164

ERV-DC8, Fe-122S and Fe-38R for ERV-DC10, Fe-58S and Fe-56R for ERV-DC14 or 165

Fe-76S and Fe-81R for ERV-DC18 using feline chromosomal DNA was directly 166

transfected to HEK293T cells and the filtered supernatant was used to infect fresh 167

HEK293T cells. Viral infection was monitored by detecting proviral ERV-DC env and 168

LTR or immunoblot analysis using anti-RD114 antibody. Viral transmission was 169

confirmed by further infection. 170

Transmission electron microscopy. HEK293T cells persistently infected with 171

ERV-DC10 were washed with D-PBS, fixed with 2% glutaraldehyde at 4°C for at least 172

2 h, washed with D-PBS (2 × 15 min) and post-fixed with 2% osmium tetroxide for 1.5 173

h. Cells were dehydrated through a graded ethanol series and propylene oxide, then 174

embedded in EPON 812 for 48 h at 60°C. Ultrathin sections were stained with uranyl 175

acetate and lead citrate, and examined with an electron microscope (JEM-1200EX; 176

JEOL, Japan). 177

178

Viral interference assays. GPLac cells (3 × 106) were transfected with each 179

env-expressing plasmid to produce pseudotype viruses. After culturing with 200 μg/ml 180


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zeocin and 1 μg/ml puromycin for >2 weeks, supernatants were collected, filtered 181

through a 0.22-μm filter and stored at -80°C. Target cells (105) were infected with viral 182

supernatant in the presence of polybrene (2 μg/ml) for 2 days, then stained with X-gal 183

(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Single-cycle infectivity was 184

titrated by counting blue-stained nuclei using a microscope following incubation with 185

X-gal. X-gal-positive cells were considered infectious units. 186

187

Fluorescence in situ hybridization (FISH) analysis. PBMC cultures stimulated with 188

Concanavalin A were treated with 5-bromo-2′-deoxyuridine for 5.5 h and harvested 189

after colcemid treatment for 0.5 h. Replication-banded chromosomes were obtained by 190

exposure of the chromosome slides to UV light after staining with Hoechst 33258. The 191

pTKDC10L2.8 (2.8 kb of ERV-DC10 flanking sequences by PCR-amplified with 192

primers, Fe-137S (5'-ATTCCTTAGCTCATGGTCCTTTTCT-3') and Fe-38R 193

(5'-CACACATGCTCTAGACACAATACCC-3')) and pTKDC18L3.4 (3.4 kb of 194

ERV-DC18 flanking sequences by PCR-amplified with primers, Fe-146S 195

(5'-GTCAATTGGACTGCCTTGAATCTAT-3') and Fe-138R 196

(5'-AAAGTTCCCAGATAATCCTGATGGT-3')) plasmid DNAs were labelled with 197

digoxigenin-11-dUTP by nick translation for use as probes. After hybridization, the 198

slides were washed and probe signals were detected with Cy3-labelled anti-digoxigenin. 199

FISH images were captured with the CW4000 FISH application program (Leica 200

Microsystem, Wetzlar, Germany). 201

Phylogenetic and genomic analyses 202

Nucleotide sequences of ERV-DC regions and amino acid sequences of Pol and Env 203


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used in the phylogenetic analyses are summarized in Supplementary Table 5. For these 204

analyses, we generated multiple alignments for each gene using MAFFT (L-INS-i)(16). 205

Amino acid substitution models of LG (20) with gamma-distributed rate variation (+Γ, 206

α = 1.672557) and rtREV(8) with gamma-distributed rate variation and inferred 207

proportion of invariable sites (+Γ+I, α = 0.912332, invar = 0.000117) were selected for 208

Env and Pol alignments, respectively, using the Akaike information criterion 209

implemented in PROTTEST 3 (6). Phylogenetic trees were constructed using the 210

maximum-likelihood method (12) in RAxML v7.2.8 with robustness evaluated by rapid 211

bootstrapping (1000 times). To identify ERVs we used RetroTector©(35) and default 212

parameters for genomes of various species provided by Ensembl 213

(http://www.ensembl.org/). Four types of retroviral-like sequences in the genome were 214

identified and we conducted a homology search with BLASTP using the Pol or Env 215

sequences of ERV-DC as a query. 216

Time estimation of ERV-DC insertion. Because the mutation rate of LTRs in 217

ERV-DCs is unknown, we alternatively utilized a mean divergence rate of non-coding 218

regions in the domestic cat genome (1.2 × 10–8 substitutions/site/year)(21). Genetic 219

distances between LTRs were calculated using the Kimura two-parameter model (17). 220

221


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Results 222

Characterization of endogenous gammaretroviruses in the domestic cat 223

(ERV-DCs) 224

Ten full-length and three partial feline endogenous gammaretroviruses, which were 225

previously partially characterized as ECE1 and FcEV (3,41), were isolated by genomic 226

library screening and PCR using locus-specific primers. Sequence analysis revealed that 227

four of these proviruses (ERV-DC8, DC10, DC14 and DC18) have intact ORFs for gag, 228

pol and env (Fig. 1) and could potentially produce infectious retroviruses. Although 229

ERV-DC10 and 18 were isolated from different cats and had different integration loci, 230

their ERV-DC nucleotide sequences were almost identical. Only 1 bp out of 8863 in the 231

primer binding site was different between ERV-DC10 and 18. The gag gene had a 232

predicted size of 1644 bp and encoded a putative 547-aa protein. The pol gene had a 233

predicted size of 3570 bp and the deduced Pol protein encoded a putative protease, 234

reverse transcriptase, RNaseH and integrase. It is likely that the Gag and Pol proteins 235

are synthesized as a single large polypeptide precursor via termination suppression. The 236

env ORF of ERV-DC10/18 was located in a different reading frame than that of the 237

gag–pol gene. The env gene had a predicted size of 2016 bp and encoded a putative 238

671-aa protein, including a signal peptide, surface (SU) protein and transmembrane 239

(TM) protein. 240

Insertional polymorphic distribution of ERV-DC in cats 241

Multiple bands of ERV-DCs were detected in the healthy domestic cat genome by 242

Southern blot analysis with a specific env probe (Fig. 2A), indicating that ERV-DCs 243


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have infected cats and are vertically inherited. An insertional polymorphic distribution 244

of ERV-DC was determined among 244 cats in Japan using PCR primers based on the 245

genomic DNA flanking sequences unique to each ERV-DC, or with second primers 246

based on the proviral sequence (Fig. 2B). Randomly chosen cats from different genetic 247

backgrounds and geographic origins within Japan were screened for the presence of 10 248

full-length ERV-DC proviruses and three partial ERV-DC proviruses. All were 249

represented in the domestic cat genome at a frequency of 0.4–100% (Fig. 2C and Table 250

S1-1). We estimated that each cat had 7–17 copies of ERV-DC proviruses, with the 251

majority having 9 copies. The ERV-DC10 locus was heterozygous in 29.5% of the cats 252

examined and homozygous in 8.2%. The ERV-DC18 locus was detected only in one 253

cat (cat ON-T) and was heterozygous. When we examined ERV-DC provirus in siblings 254

of ON-T, the ERV-DC18 locus was heterozygous in three of four siblings which were 255

born to the same mother. Interestingly, these 3 cats also had ERV-DC10 and tested 256

positive for FeLV infection (Fig. 2D and Table S1-2). The sequences of ERV-DC18 are 257

almost identical to those of ERV-DC10; therefore, ERV-DC10 appears to have been 258

mobilized as an infectious virus in one of the parents of the three positive siblings, 259

generating provirus ERV-DC18. Although some ERV-DCs have become fixed in the cat 260

genome, others have not (Fig.2C). These results indicate that ERV-DC proviruses are 261

currently invading domestic cats. Sequence analysis verified that ERV-DC sequences 262

are integrated into the cat genome, and analysis of genomic sequences flanking the 263

proviruses, including identical target site duplication (TSD), identified unequivocally 264

the various ERV-DCs (Fig.1). Furthermore, 5′ and 3′ LTR sequences of each 265

ERV-DC were found to have high sequence identity or were identical with each other 266

(Table 1). There was no evidence of LTR recombination as determined by analyzing the 267


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4-bp identical TSD sequences (Fig. 1) and the phylognetic tree of LTR sequences 268

(Fig.5D). These results suggest that integration of ERV-DC occurred relatively 269

recently in the evolutionary history of cats. Supporting this idea is the absence of 270

ERV-DC proviruses and RD-114 in the Tsushima wild cat Prionailurus bengalensis 271

euptilurus (Fig. 2A and Fig. S1), which lives in close proximity to domestic cats. The 272

domestic cat is estimated to have separated from the leopard cat and the Tsushima wild 273

cat approximately 6.2–9.3 million years ago (MYA)(10,32). By comparing the 274

nucleotide sequences of the 5′- and 3′-LTR of ERV-DC7 (Table 1), which is fixed in the 275

cat population (Fig. 2C and Table S1-1), we estimated that the oldest time of ERV-DC 276

insertion was 2.8 MYA, assuming an average divergence rate of non-coding regions of 277

domestic cat genomes (21). Thus, we hypothesize that ERV-DC and RD-114 infected 278

domestic cats after separation from the leopard lineage. 279

280

Infectious viruses, ERV-DC10 and ERV-DC18 281

Since ERV-DC8, -DC10, -DC14 and -DC18 have intact gag/pol/env reading frames, we 282

carried out studies to determine if they were infectious. We transfected human 283

embryonic kidney 293T (HEK293T) cells with proviral plasmid DNA, and ~72 h later, 284

collected 0.22-μm-filtered cell-free supernatants that were used to infect HEK293T and 285

HeLa cells. ERV-DC10 and -DC18 were shown to be infectious by the presence of 286

proviral DNA and viral RNA (Fig. 3A,B), whereas ERV-DC8 and -DC14 were not. 287

Viral Gag and Env proteins were detected by immunoblotting (Fig. 3C). Surprisingly, 288

goat anti-FeLV gp70 detected ERV-DC Env as well as FeLV Env. Human cell lines 289

persistently infected with ERV-DC10 and -DC18 were established. Cell-free 290


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supernatants from HEK293T cells infected with ERV-DC10 and -DC18 could infect a 291

number of other human cancer cell lines, such as K562, Molt-4, HepG2, THP-1, dog 292

mammary tumour cells (KwDM) and monkey Vero cells (Fig. 3D and Fig. S2). The 293

viruses were also able to infect the feline kidney cell line CRFK, but not other feline 294

cell lines that can be infected with other feline retroviruses (e.g., AH927 fibroblasts that 295

are readily infected with FeLV; G355 astrocytes that can be infected with RD-114). 296

Transmission electron microscopy of HEK293T and HeLa cells persistently infected 297

with ERV-DC10 revealed 90–100-nm particles that were morphologically consistent 298

with a type C retrovirus (Fig. 3E). 299

We developed an alternative method to determine whether ERV-DC proviruses in 300

cat DNA are infectious. Full-length ERV-DC10 or ERV-DC18 proviral DNA from 301

primary cat PBMC was amplified by PCR with locus-specific primers and the PCR 302

products—without cloning of proviruses—were transfected into HEK293T cells. After 303

2-3 weeks, each filtered supernatant was used to infect HEK293T cells. As a result, 304

ERV-DC10 or -18 were shown to persistently produce infectious virus (Fig. 4A, B). 305

Even HEK293T cells transfected with DNA from the AH927 feline cell line, which has 306

a homozygous ERV-DC10 locus, produced infectious virus (Fig. 4A). Virus production 307

from DNA from cats that lacked ERV-DC10 or ERV-DC18 was never seen in cultures 308

(Fig. 4C), indicating that this method specifically assesses the activity of ERV-DC10 309

and -DC18 loci. Using this method, we also found that DNA from cats harbouring 310

ERV-DC8 and ERV-DC14 proviruses was unable to produce infectious virus (Fig. 4C), 311

consistent with data obtained using molecular clones of ERV-DC8 and -DC14. These 312

results demonstrate that ERV-DC10 and ERV-DC18 are potentially active and their 313

genomes encode infectious viruses. 314


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To define the active locus, we mapped two active loci by fluorescence in situ 315

hybridization (FISH) analysis: ERV-DC10 mapped to feline chromosome C1q12-21 and 316

ERV-DC18 to feline chromosome D4q14 (Fig. 4D,E). These results indicate that 317

ERV-DC10 and -DC18 loci are distinct from each other and we speculate that the 318

ERV-DC10 provirus on C1q12-21 mobilized to D4q14. We could not isolate ERV-DC10 319

viruses when PBMC from ERV-DC10–positive cats were cultured with HEK293T cells. 320

Thus, although infectious ERV-DC10 provirus is present in the genome of domestic cats, 321

PBMC and AH927 cells may not produce infectious viruses or ERV-DC10 may be 322

silenced in these cells. 323

Phylogenetic analyses of ERV-DCs 324

Phylogenetic analyses indicated that ERV-DC proviruses were genetically distinct from 325

exFeLVs, enFeLVs and RD-114, and comprised a separate group of gammaretroviruses 326

(Fig. 5A, B). ERV-DC proviruses obtained in this study were classified into three 327

groups based on their env genes (Fig. 5C). This classification was supported by the 328

phylogenetic tree of their LTR sequences (Fig. 5D), and suggests that independent 329

invasion of ERV-DCs may have occurred in the ancestry of domestic cats. 330

To examine if ERV-DCs interact with DNA of other species, we extracted 331

ERV-DC-like ERVs from the genome data of 12 different species (Table S2). As a result, 332

22, 19, 3, and 2 ERV loci were detected in the genomes of the house mouse, brown rat, 333

common chimpanzee and rhesus macaque, respectively (Table S3), although no 334

orthologous relationships were found among them. We then conducted phylogenetic 335

analyses of ERV-DCs using these ERV elements with various endogenous or exogenous 336

viruses (Fig. 5A, B). The Pol protein of ERV-DC was most closely related to that of 337


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RD-114 and BaEV (Fig. 5A), whereas the Env/TM protein of ERV-DC was more 338

closely related to that of the FeLV family (Fig. 5B). RD-114 is genetically related to 339

BaEV and both of these viruses are recombinant viruses that contain a type C 340

(gammaretrovirus group) ORF for gag-pol and a type D (betaretrovirus group) env gene, 341

and supposedly arose from cross-species transmission (1,15,36). The amino acid 342

identity was 98.6% for Pol proteins of ERV-DC18 and RD-114 SC3C, and 81.1% for 343

the TM regions of Env proteins of ERV-DC18 and FeLV-A clone 61E (55.8% for the 344

entire protein). In addition, the ERV sequences in the house mouse, brown rat, common 345

chimpanzee and rhesus macaque genomes were also found to be similar to ERV-DCs 346

(Fig. 5A, B). Pol from ERV-DC10/DC18 is most highly related to the Pol of 347

endogenous primate viruses, including chimpanzee, rhesus macaque, and baboon. In 348

contrast, Env/TM from ERV-DC10/18 is closely related to FeLV and rodent retroviruses. 349

These results indicate that ERV-DCs should be classified as a strain of 350

gammaretroviruses, and that ERV-DCs or ERV-DC-related viruses have passed through 351

several species. The lack of ERV-DC-like elements in the genomes of species other than 352

primates and rodents suggests that ERV-DCs have spread through interspecies 353

transmission among rodents, primates and cats. The interspecies transmission of 354

ERV-DCs may make it possible to generate recombinant viruses, and ERV-DCs could 355

be donor and/or acceptor viruses in domestic cats. 356

357

Evidence for transduction of ERV-DC env into FeLV 358

Recombination events occur in retroviruses by template switching during viral reverse 359

transcription (7). We discovered from the spleen of an FeLV-positive cat (ON-T; Fig. 360


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6A) a recombinant retrovirus, designated FeLV-D (strain FeLV-D/ON-T), containing the 361

env gene of ERV-DC and LTR, gag and pol sequences from FeLV. Sequence analysis 362

demonstrated that the env gene of FeLV-D was derived from ERV-DCs (Fig. S3A) and 363

the LTR of FeLV-D was derived from FeLV (Fig. S3B). The gag–pol gene contained an 364

ORF but also some deletions (Fig. 6A), suggesting that the virus is replication defective. 365

To determine if FeLV-D is expressed in the cat, spleen RNA from the cat ON-T was 366

amplified by RT-PCR using FeLV-D–specific primer pairs corresponding to FeLV 367

5′LTR and ERV-DC env sequences. Major bands corresponding to the genomic RNA 368

and spliced env mRNA were detected (Fig. S4) and confirmed by sequencing. To 369

determine if FeLV-D could be isolated from cells cultured from the spleen of cat ON-T, 370

we homogenized the spleen, filtered the sample using a 0.22-μm filter and used the 371

homogenate to inoculate HEK293T cells. FeLV-D, as well as prototype FeLV, was 372

detected at 4 weeks post-inoculation by PCR (Fig. 6B). The FeLV-D/ON-T strain was 373

successfully isolated in association with prototype FeLV, and FeLV-D was packaged as 374

a retrovirus. The supernatant from HEK293T cells infected with FeLV-D/ON-T for 6 375

weeks was used to inoculate fresh HEK293T and HeLa cells, and the virus was 376

successfully transmitted between the cells in culture. We re-cloned FeLV-D/ON-T 377

(designated FeLV-D/re-T) from the chromosomal DNA of HEK293T cells persistently 378

infected with FeLV-D and verified that the sequences of FeLV-D/re-T and 379

FeLV-D/ON-T are identical (Fig. S3A). When the DNA from peripheral blood or 380

tumour tissues from 283 other FeLV-positive cats was screened for the presence of 381

FeLV-D, three positive samples were detected (Fig. 6C). One cat (ON-C) was a sibling 382

of ON-T. FeLV-D was molecularly cloned from the three other FeLV-D–positive cats 383

(FeLV-D/ON-C, FeLV-D/Ty26 and FeLV-D/44B) in the region of env and 3′LTR (Fig. 384


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S3A,B,C). A schematic representation of the structures of the env genes of the various 385

FeLV-Ds is shown in Fig. 6D. Thus, FeLV-D appears to have been generated by 386

recombination between FeLV and ERV-DC, indicating that ERV-DC can serve as a 387

donor and/or acceptor in the generation of recombinant viruses. The recombination 388

junction between ERV-DC and exFeLV varied among FeLV-D strains, suggesting that 389

FeLV-D was generated de novo in each cat. It is not clear whether FeLV-D is an 390

infectious virus by itself or whether it is pathogenic. To determine if the FeLV-D env 391

gene plays a role in infection, FeLV-D env-expressing pseudoviruses were generated 392

that could transmit β-galactosidase (Fig. 7). The FeLV-D env-expressing pseudotype 393

viruses from FeLV-D/ON-T and FeLV-D/Ty26 could infect cells already infected with 394

FeLV-A, B or C, or cells persistently infected with ERV-DC10. Therefore, our data 395

show that FeLV-D belongs to a different interference group than ERV-DC10. These 396

results indicate that the receptor usage for both FeLV-D and ERV-DC10 to enter cells is 397

distinct from FeLV A, B, and C. 398

Discussion 399

This study characterizes 13 endogenous retroviral proviruses in the cat belonging 400

to a class of ERVs, ERV-DC, that has previously been only partially characterized. We 401

show that two of these ERV-DCs, ERV-DC10 and ERV-DC18, are novel feline 402

infectious gammaretroviruses that are inherited on chromosome C1q12-21 and D4q14, 403

respectively. Thus, our study is the first to demonstrate the existence of endogenous, 404

infectious retroviruses in the genome of the domestic cat. Approximately 38% of the 405

cats examined in Japan have heterozygous or homozygous ERV-DC10 loci. Unlike 406

ERV-DC7, which is fixed in the cat genome but contains deleterious mutations that 407


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make it non-infectious, ERV-DC10 has not yet been fixed in the cat genome. It is not 408

known whether ERV-DC10 will eventually disappear or become fixed in cats. Although 409

the open reading frames of ERV-DC8 and ERV-DC14 are intact, they are unable to 410

make infectious viral particles, most likely due to point mutations. It is unclear 411

whether ERV-DCs could be harmful or beneficial to the host. However, previous studies 412

have indicated that the presence of other ERVs in vertebrates is associated with 413

protection against infection of related exogenous retroviruses as well as with the 414

generation of pathogenic viruses or tumors (9,11,26). 415

Our studies further indicate that ERV-DC10 was not able to produce infectious 416

particles when it was present in some cell types, including feline PBMCs (ERV-DC10 417

+/+ genotype) and the feline fibroblast cell line AH927 (ERV-DC10 +/+), suggesting 418

that the ERV-DC10 promoter may be modified in a tissue-specific manner, similar to the 419

endogenous human ERV syncytin (24), as expression of retroviral genes can be 420

controlled by transcriptional modification (43). Moreover, an infectious provirus not 421

being expressed may be related to its methylation status in the cell, a common 422

mechanism by which endogenous retroviruses are silenced (22). Further investigation 423

will be necessary to understand the gene sequences in ERV-DCs controlling expression 424

and viral production. 425

Endogenous retroviral sequences have been known to exist in the domestic cat, 426

but until now they have not been identified as encoding infectious viruses. Although 427

RD-114, which was generated by xenotransplantation, is described as an infectious 428

feline ERV (23), an infectious RD-114 provirus has never been detected in the genome 429

of domestic cats. Recent work shows that novel, infectious mouse retroviruses can be 430


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generated through in vivo passage of human cells in rodents (30). It is, therefore, 431

possible to generate an infectious ERV through in vivo passage in other species. 432

Our phylogenetic analyses revealed that the pol gene of ERV-DCs has high 433

similarity to those of BaEV, RD-114 and primate ERVs, whereas the env/TM gene of 434

ERV-DCs is related to those of FeLV, MuLV and rodent ERVs. The fact that infectious 435

ERV-DCs are present in domestic cats and ERV-DC-like elements are detected in cats, 436

rodents and primates suggests that ERV-DCs may have been transmitted to primates or 437

rodents, or vice versa, becoming part of the DNA of the infected species. 438

FeLV-D was detected and characterized in this study as a recombinant virus 439

generated by transduction of the ERV-DC env gene into FeLV. This discovery supports 440

the idea that ERV-DCs may act as donor and/or acceptor viruses to generate new 441

recombinant viruses. We showed that FeLV-D belongs to a different interference group 442

from that of ERV-DC10, consistent with the fact that ERV-DC10 belongs to ERV-DC 443

group III, whereas the env genes in all 4 FeLV-D strains are similar to ERV-DC group I. 444

ERVs belonging to groups I and III may use distinct cell surface receptors and it will be 445

important to characterize these receptors and assess infectivity of the ERV-DC 446

recombinant viruses. 447

ERV-DC is present in the cat genome with different copy numbers and insertional 448

polymorphisms. Reinfection or infection with ERV-DCs may vary among individual 449

cats based on whether the host genes permit or restrict infection. ERV-DCs are present 450

in domestic cats, but not in the Japanese Tsushima leopard cat, Prionailurus bengalensis 451

euptilurus. Based on estimation of viral integration time and ERV-DC LTR sequences, 452

ERV-DCs infected domestic cats within the past 2.8 million years, after separation from 453


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the leopard lineage. It will be interesting to determine whether ERV-DC proviruses have 454

played a role in the evolution of cats or in the generation of polymorphisms in the cat 455

genome. 456

In conclusion, our findings provide evidence for the existence of infectious 457

ERV-DCs in the domestic cat. Because these ERV-DCs are currently being transmitted 458

among cats, and perhaps other species, as infectious ERVs or as recombinant viruses 459

containing ERV-DC sequences, future studies should follow their evolution and assess 460

the consequences of their presence for the health of domestic cats and other species. 461

462

463

464

465

466

467

468

469

470

471

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473

Data deposition The sequences reported here have been deposited in the 474

DDBJ/EMBL/GenBank and the accession numbers are AB673426-AB673432, 475

AB674572-AB674577 and AB674439-AB674452. 476

Acknowledgements We thank Drs. T. Kitamura and M. Nakaya for providing 477

packaging cells and expression plasmids. We also thank Drs. N. Oishi (Oishi Veterinary 478

Hospital) and H. Yamamoto (Tsushima Wildlife Conservation Center) for providing 479

blood and tissue samples. We are grateful to Sandra Ruscetti (National Cancer Institute, 480

Frederick) and Kenji Baba (Yamaguchi University) for helpful discussions with the 481

manuscript. This study was supported by grants from the Ministry of Education, 482

Culture, Sports, Science and Technology of Japan (K.N.). Y.A.,H.O. and S.W. 483

contributed equally to this work. 484

485

486


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Figure legends 647

Fig. 1. ERV-DC structures. Structures of the genomes of 10 full length ERV-DC 648

proviruses and three partial ERV-DC proviruses. The gag, pol and env genes are 649

illustrated together with the 5′ and 3′ LTRs and positions of the gag and env 650

translational initiation codons (ATG). Asterisks indicate conserved stop codons. Gag 651

and Pol proteins may be synthesized as a large single polypeptide precursor via 652

termination suppression (44). Open triangle indicates a deletion of nucleotides and filled 653

triangle indicates an insertion of nucleotides. Flanking 4-bp TSD sequences are shown 654

for each provirus. 655

Fig. 2. Detection of ERV-DC proviruses in the domestic cat genome. (A) Southern 656

blotting of ERV-DC with an env probe derived from FeLV-D/ON-T in chromosomal 657

DNA from the domestic cat (F.s. catus) and P.b. euptilurus. (B) PCR for detection of 658

pre-integration sites (PISs) and proviruses. Red boxes: viral LTRs; yellow boxes: target 659

site duplications. Primers A–D are shown. The AC primer pair generated a PIS and/or 660

full-length provirus. (C) Insertional polymorphisms of 13 ERV-DCs in 244 domestic 661

cats. Green: provirus was detected on heterozygous loci; Red: provirus was detected on 662

homozygous loci; Yellow: provirus was detected (D) ERV-DC18 was only present in 3 663

of 4 siblings from one family. Each PIS, full-length ERV-DC10 or ERV-DC18 was 664

detected by primers Fe-122S and Fe-38R for ERV-DC10 or Fe-76S and Fe-81R for 665

ERV-DC18 (Table S4). 666

Fig. 3. ERV-DC10 and ERV-DC18 are infectious viruses. (A) env and pol were detected 667

by PCR at 2 and 8 days after infection of HEK293T cells. The supernatants from 8-day 668

cultures were used to infect HeLa cells. M: mock infection; 10: infected with 669


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ERV-DC10; 18: infected with ERV-DC18. (B) Viruses collected from supernatants were 670

used to detect env and pol genes by RT-PCR. (C) ERV-DC10 and ERV-DC18 proteins 671

were detected in HEK293T cells using goat antiserum against disrupted RD-114 virions 672

and anti-FeLV gp70 antibody. Anti-RD114 antibodies recognize many ERV-DC proteins, 673

indicated by arrows, but not ERV-DC Env. (D) Susceptibility of cells to infection by 674

ERV-DC10/ERV-DC18. (E) Transmission electron microscopy of retroviral particles in 675

HEK293T cells persistently infected with ERV-DC10. The bar under each panel 676

represents a 100-nm scale. 677

Fig. 4. Active loci of ERV-DC10 and ERV-DC18. (A) ERV-DC10; (B) ERV-DC18 or 678

(C) ERV-DC10, -DC8 or -DC14 DNA amplified by PCR with locus-specific primers 679

using feline chromosomal DNA was directly transfected to HEK293T cells and the 680

filtrated supernatant was used to infect fresh HEK293T cells. Viral infection was 681

monitored by detecting proviral DNAs or viral proteins. Each proviral genotype is 682

indicated as -/- (wild), +/- (heterozygous) and +/+ (homozygous). Independent DNA 683

samples were used in the figures. AH927 is a feline cell line with ERV-DC10 +/+. (D 684

and E) FISH analysis of ERV-DC10 and ERV-DC18 loci on feline chromosomes. 685

Fig. 5. Maximum-likelihood phylogenetic trees of Pol and Env TM proteins. We used 686

amino acid sequences of entire regions of (A) Pol proteins and (B) TM subunits of Env 687

proteins for 46 or 56 endogenous/exogenous viruses (Table S5). A monophyletic subtree 688

of the ERV elements obtained from the same species was compressed into a single 689

branch as indicated by arrows. Viral origin is indicated as blue (feline), orange (primate), 690

green (rodent), gray (avian) or black (other species). The percent values were 691

determined from 1000 repeats of fast bootstrapping using RAxML (37) and are 692


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indicated at the branch junctions. More than 70 bootstrap values are shown. 693

Maximum-likelihood phylogenic trees of ERV-DC env and LTR are indicated in Fig.5C 694

and Fig.5D. Nucleotide sequences of (C) entire ERV-DC env regions or (D) 5′ and 695

3′ LTRs in ERV-DCs obtained were used. The general time-reversible model with rate 696

heterogeneity among sites (GTR+Γ) was utilized. The percent values were determined 697

from 1000 repeats of fast bootstrapping using RAxML and are indicated at the branch 698

junctions. 3 or 5 indicates 3’ LTR or 5’ LTR. ECE1 (Accession No. X51929), Fc21 699

(AF155060), Fc41 (AF155061), SC3C (EU030001), CRT (AB559882) 700

701

Fig. 6. Transduction of ERV-DC env into FeLV. (A) Schematic representation of the 702

structure of FeLV-D provirus integrated in spleen DNA of cat ON-T and FeLV-A/61E. 703

White indicates FeLV sequences and gray indicates ERV-DC sequences. (B) Isolation of 704

FeLV-D in HEK293T cells inoculated with spleen extracts from cat ON-T. FeLV-D- and 705

FeLV env-specific PCR was carried out. (C) A total of 283 DNA samples were screened 706

for the presence of FeLV-D and four positive samples were detected. (D) Structure of 707

FeLV-D env. Recombination pattern of each FeLV-D env is indicated. White indicates 708

FeLV sequences and gray indicates ERV-DC sequences. 709

Fig. 7. Infection assay of LacZ pseudotype virus. FeLV-A/Glasgow-1 env, 710

FeLV-B/Gardner-Arnstein env, FeLV-C/Sarma env, FeLV-D/ON-T env, FeLV-D/Ty26 711

env and ERV-DC10 env were used for each pseudotype virus preparation. HEK293T 712

cells infected with FeLV-A/clone33, FeLV-B/Gardner-Arnstein, FeLV-C/Sarma, 713

ERV-DC10 or both FeLV-A/clone33 and FeLV-D/ON-T were used as target cells for an 714

interference assay. X-gal-positive cells were counted as infectious units (I.U.). Arrow 715


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indicates viral interference. 716

717

718

719

720


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ERV-DC1 ERV-DC2 ERV-DC3 ERV-DC4 ERV-DC7 ERV-DC8 ERV-DC10 ERV-DC14 ERV-DC17 ERV-DC18 ERV-DC19

0 3 1 0 42 1 0 1 4 0 3

100 99.5 99.8 100 96.7 99.8 100 99.8 99.5 100 99.5

Provirus Number of

Differences between 5’ and 3’ LTRs

Homology between 5’ and 3’ LTRs (%)

Length of LTR (bp) 3’LTR 5’LTR

550 555 550 550 545 550 551 551 551 551 555

550 555 550 550 566 550 551 551 550 551 555

Table 1

Properties of ERV-DC 5′ and 3′ LTRs are summarized.


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Figure 1

ERV-DC1

*5’LTR

ATG * *ATG

3’LTRgag Δpol Δenvgag - Δpol Δenv

GTGG GTGG

250bp deletion1bp deletione

5’LTRATG *ATG

3’LTRenvenv

gag( ? )ERV-DC2

*ATG**3’LTR5’LTR

ATG

Δgag pol ΔenvΔ gagERV-DC3

GGAC GGAC

TAAC TAAC

1bp insertion71bp deletion

Δ gag Δenv

* *ATG*3’LTR

1bp insertion

5’LTRATG

gag pol Δenvgag - pol Δenv

ERV-DC4

*3’LTR

ATG

envERV DC6

TGAC TGAC

GGAG

* *ATG*3’LTR

1bp deletion

1bp insertion

5’LTRATG

Δgag Δpol ΔenvΔgag Δenv

GTG

1bp insertionERV-DC7

* *ATG*3’LTR5’LTR

ATG

gag pol env

envERV-DC6

ACAA ACAA

GGAG

3 LTR5 LTR gag pol envgag - pol env

ERV-DC8


ATG

gag pol envgag - pol env

ERV-DC10

* *ATG*ATG

ATAC ATAC

TTGG TTGG

3’LTR5’LTR gag pol envgag - pol env

ERV-DC14

Δenv

*3’LTR

ATG

ΔenvERV-DC16

TATT TATT

AACC


ATG

gag pol envgag - pol env

ERV-DC18

3’LTR*

1bp insertion

5’LTRATG * *ATG

gag Δpol envgag - Δpol env

ERV-DC17 AAAC AAAC

CATA CATA

3’LTR

2.4kbp deletion

5’LTRATG * *ATG

Δgag envΔgag - Δpol env

ΔpolERV-DC19 ATTG ATTG

1kb


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Figure 2

AC

kbp100

23

9.4

6 6

+/-+/++30

405060708090100

ts w

ith p

rovi

rus

6.6

2 3

+

0102030

1 2 3 4 6 7 8 10 14 16 17 18 19

% o

f cat

ERV-DC locus no.2.3

BamHIEcoRI

B D4bp

APIS

siblings

5’LTR 3’LTRgag pol env

IntegrationC

ERV-DC provirus B

PIS(ERV-DC18)

ERV-DC18

ERV-DC10CDC

p

A

C

PIS(ERV-DC10)

c-myc


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ACFigure 3 kDa

100

M 10 18

M 10 18 M 10 18 M 10 18HEK293T (2days) HEK293T (8days) HeLa (7days)

A

50

Anti-RD114 virus

M 10 18 M 10 18 M 10 18

env

pol

F LV

293T

/FeL

V-

BAnti-FeLV gp70β-actin

FeLV

HEK293T (2days) HEK293T (8days) HeLa (10days)B

Cell lines (species) ERV-DC10/18

Molt-4 (Human) ＋

K562 (Human) ＋

DAnti-β-actin

( y ) ( y ) ( y )

env

RT + + + - - - + + + - - - + + + - - -M 10 18 M 10 18 M 10 18 M 10 18 M 10 18 M 10 18

E

K562 (Human) ＋

HepG2 (Human) ＋

HeLa (Human) ＋

HEK293T (Human) ＋

MCF7 (Human) －

THP 1 (Human) ＋

pol

THP-1 (Human) ＋

CRFK (Cat) ＋

AH927 (Cat) －

G355 (Cat) －

Vero (Monkey) ＋

C 7 (M k )Cos7 (Monkey) －

NIH3T3 (Mouse) －

L929 (Mouse) －

BHK21 (Hamster) －

KwDM (Dog) ＋


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DAFigure 4

C -/- -/- +/- +/- +/+ +/+ AH927

env

ERV-DC10

pLTR

β-actinkDa75

q

50

WB:Anti-RD114

C1ERV-DC10 locus(C1q12-21)

E37

β-actin

pB C -/- -/- -/- -/- +/- +/- +/-

ERV-DC18

env

LTR

q

C DC8 DC14 DC10ERV-DC10

LTR

β-actin

D4ERV-DC18 locus(D4q14)

C -/- -/- -/- -/- +/- +/- +/+env

β-actin


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A

Figure 5

PolB

Env/TM FERV-18100 ERV-DC18FERV-1876 ERV-DC18Pol Env/TM FERV-ECE1

FeLV-A clone33

FeLV-A 61E

enFeLV AGTT

Rat ERVs96

100

8581

ERV-DC ECE1

viru

s

FERV-ECE1

RD114 SC3C

BaEV M7

Macaque ERVs

Chimpanzee ERVs100

7699

9999

99

ERV-DC ECE1

Mouse ERVs and XMRV

Mouse ERV Chr.18

F-MuLV

M-MuLV

PERV C

Gamma

77

77

96

87

Gam

mar

etro

v

FeLV-A clone33

FeLV-A 61E

enFeLV AGTT

Rat ERVs

F-MuLV

Gamma

100

9099

87

100

8781 mar

etro

viru

s

PERV-C

KoRV

GaLV

BaEV M7

RD114 SC3C

REV-A Beta

82

95

89

viru

s

M-MuLV

XMRV

Mouse ERVs

PERV-C

KoRV

GaLV10090

100

88

100

Gam

m

MPMV

SRV-4

Beta

JSRV

HERV-K

MMTV

Beta

99

100

95

usB

etar

etro

vGaLV

REV-A

HERV-K

SRV-4

MPMV

JRSVBeta

998587

100

Bet

a-re

trovi

rus

JSRV ALV

MAVAlpha

100

0.5

Alph

a-re

trovi

r u MMTV

ALV

MAVAlpha

87

100

0.5

rAl

pha-

retro

viru

s


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Figure 5

ERV DC3 5

env LTRDC

ERV DC1

ERV-DC3 5

ERV-DC1 5

ERV-DC1 3

ERV-DC4 5

ERV-DC4 378

39

38 ERV-DC1

ERV-DC8

ERV-DC14

ERV-DC17

ERV-DC3

ERV-DC4Group I

100

98

ERV-DC14 5

ERV-DC14 3

ERV-DC3 3

ERV-DC17 5

ERV-DC17 3

62

38

50

54

98Group I

Group I

ERV DC4

Fc21

ERV-DC5

ERV-DC2

ERV-DC19

ERV-DC7

91

100

100

ERV-DC8 3

ERV-DC8 5

ERV-DC19 5

ERV-DC19 3

ERV-DC7 3

100

81

Fc41

ERV-DC16

Group II

ECE1

ERV-DC6

ERV-DC10

ERV DC18

Group III

100100

100

100

ERV-DC16 3

ERV-DC7 5

ERV-DC6 3

ECE1 3LTR

ERV-DC18 5

8497

95

Group III

Group II Group II

ERV-DC18100

0.01

ERV-DC10 3

ERV-DC10 5

ERV-DC18 3

SC3C 5LTR

CRT1 5LTR

100

100

95

RD114

Group III

CRT1 5LTR100

0.01


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Figure 6

A 5’LTR 3’LTRgag ATG env ATGpol stop env stopgag stop* **

gag - polFeLV-A/61E

FeLV-D/ON-T

envgag pol

1kb

gag stop5’LTR 3’LTRgag ATG env ATGpol stop env stop

env

* **

gag - Δpol

B FeLV-D positiveC 1632bp 720bp

FeLV-D env0 3 4 5 6

wks

c-myc

FeLV-D envβ-actinFeLV env

D FeLV-A/61E

F LV D/ON T

3’LTRSU TM

FeLV-D/ON-T

FeLV-D/re-T

FeLV-D/ON-C

FeLV-D/Ty26

FeLV-D/44B

ERV-DC8


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A (Glasgow1)

B

HEK293T/FeLV-A (clone33)

A (Glasgow1)

B

HEK293T/FeLV-BFigure 7

BCD (ON-T)D (Ty26)

ERV-DC10

FeLVBCD (ON-T)D (Ty26)

ERV-DC10

FeLV

ERV DC10LacZ Titer (I.U.)102 104 106

A (Glasgow1)

HEK293T/FeLV-C

ERV DC10LacZ Titer (I.U.)102 104 106

A (Glasgow1)

HEK293T/ERV-DC10

BCD (ON-T)D (Ty26)

FeLVBCD (ON-T)D (Ty26)

FeLV

ERV-DC10LacZ Titer (I.U.)102 104 106

ERV-DC10LacZ Titer (I.U.)102 104 106

HEK293T/FeLV-A (clone33)+FeLV-D (ON-T)

A (Glasgow1)

BCD (ON-T)

FeLV

D (Ty26)ERV-DC10

LacZ Titer (I.U.)102 104 106


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1 infectious endogenous retroviruses in cats and emergence of recombinant viruses yukari

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