Germ Cell Expression of an Isolated Human ... - Journal of Virology

JOURNAL OF VIROLOGY, Dec. 1999, p. 9976–9983 0022-538X/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 12

Germ Cell Expression of an Isolated Human Endogenous Retroviral Long Terminal Repeat of the HERV-K/HTDV Family in Transgenic Mice ARMELLE E. CASAU,1 JOE E. VAUGHAN,2 GUILLERMINA LOZANO,2

AND

ARNOLD J. LEVINE3*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 085441; Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 770302; and The Rockefeller University, New York, New York 10021-63993 Received 3 June 1999/Accepted 8 September 1999

In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving lacZ expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual. was first isolated by using a Syrian hamster intracisternal A particle (IAP) pol probe and was shown to have some homology to the mouse mammary tumor virus (MMTV) env gene (34). HERV-K elements entered the primate lineage after the divergence of New World monkeys and Old World monkeys more than 30 million years ago and underwent amplifications as well genomic rearrangements (40). Human-specific integration events have been reported and indicate relatively recent HERV-K activity (30). HERV-K is present in about 30 to 50 copies in the human genome (34), along with an estimated 10,000 solitary long terminal repeats (LTRs) (21). Unlike many other HERV family members, some of the HERV-K proviruses have retained open reading frames for their viral genes (13, 17, 25, 27, 28, 33, 44). Although no one provirus has been demonstrated to produce the HERV-K viral particles, an almost intact provirus with all open reading frames has been found on chromosome 7 (29). Noninfectious particles derived from as of yet unidentified HERV-K proviruses have been observed in testicular teratocarcinoma cell lines and tumors, as well as in the placenta, but not in the vast majority of other tumor types or cell lines (4, 8, 24, 39). Testicular teratocarcinomas are germ cell tumors (GCTs) that are composed of an undifferentiated embryonal carcinoma (EC) stem cell population and a variety of differentiated cell populations (1, 2). The human teratocarcinoma-derived viruses (HTDVs), which are expressed in these tumors, are arrested at late budding stages and lack the electron-lucent space between the envelope and the core shell indicative of mature particles (4). These particles seem to no longer be observed in the differentiated derivatives of GCTs such as differentiated embryonal carcinoma cells, cultures of yolk sac carcinomas,

Endogenous retroviruses are present in multiple copies dispersed throughout the genomes of host species (9, 47). These retroelements have been vertically transmitted through the germ line as Mendelian genes for millions of years and may represent the endogenous counterparts of exogenous retroviruses that infected ancestral species long ago. It is estimated that up to 10% of the human genome is composed of sequences that were reverse transcribed and that up to 1% of the human genome is made up of endogenous retroviruses that are very similar to exogenous retroviruses both in sequence and in structure (3, 5, 47). Both the prevalence and maintenance of these elements suggest that they may play a role in the biology of the host species. Endogenous retroelements may affect their hosts via multiple mechanisms (46, 47): (i) they can act in trans by expressing viral gene products that could interfere with or modulate cellular activities (20); (ii) they can act in cis by activating neighboring cellular genes with their regulatory sequences (11) or by retrotransposing and leading to insertional mutagenesis (31, 32); (iii) they can promote genomic plasticity as well as contribute to allelic variations (42, 48); (iv) and they have been shown, in some cases, to potentially play a role in autoimmune diseases such as glomerulonephritis (18, 37, 46). One human endogenous retrovirus of interest is HERV-K (human endogenous retrovirus K), where the K denotes the lysine tRNA used by this element as a primer for reverse transcriptase (35). An HERV-K family member, HERV-K10, * Corresponding author. Mailing address: Office of the President, The Rockefeller University, 1230 York Ave., New York, NY 100216399. Phone: (212) 327-8080. Fax: (212) 327-8900. E-mail: alevine @rockvax.rockefeller.edu. 9976

VOL. 73, 1999

GERM CELL EXPRESSION OF HERV-K/HTDV LTR

and teratomas (8), although it has also been suggested that HTDVs are actually budding from the trophoblastic component of differentiating teratocarcinoma cell cultures (22, 24). High levels of expression of HERV-K members appear to be restricted to GCTs (including testicular teratocarcinoma cell lines) and their testicular precursor lesions (14, 15, 23). Mature and immature teratomas and spermatocytic seminomas with no embryonal carcinoma cell component show no HERV-K expression (15). Furthermore, many other tissues, tumor types, and cell lines do not demonstrate detectable levels of HERV-K expression (15, 23). Occasionally, a low level of expression has been found in chronic myeloid leukemias (6), leukocytes (7), placenta (38), peripheral blood mononuclear cells, and brain tissues of healthy persons and multiple sclerosis patients (36). The significance of these sporadic and peculiar expression patterns remains elusive. In this study, an active HERV-K LTR has been isolated and used to study the expression patterns of the viral promoterenhancer(s) in order to determine when the transcriptional regulatory elements of a typical HERV-K virus direct gene expression. The isolated HERV-K LTR was demonstrated to drive high-level expression of a reporter gene in human and murine teratocarcinoma cell lines but not in the differentiated counterparts or in various other tumor cell lines tested. To study the expression pattern of this viral LTR in vivo, transgenic mice that harbor the HERV-K LTR upstream of the ␤-galactosidase reporter gene were generated. Both Northern and reverse transcription-PCR (RT-PCR) analyses of mouse tissues indicate that expression is limited primarily to the more undifferentiated spermatocytes of the adult testes and, to a lesser extent, the brain. These observations suggest that some HERV-K members harboring similarly active LTRs might be developmentally regulated, being active primarily in adult gonocytes and in undifferentiated GCTs, but not in their differentiated counterparts nor in a variety of other tissue and tumor types. MATERIALS AND METHODS Sequences. The accession number for the 5⬘ LTR of HERV-K10 is M12851. The accession number for HERV-K LTRE3 is AF148679. The BLAST program from NCBI was used to compare the two LTRs. Cell lines. The cell lines used were Tera 1 (human teratocarcinoma ATCC HTB 105), Tera 2 (human teratocarcinoma ATCC HTB 106), 1218E (human teratocarcinoma cells obtained from P. A. Andrews), F9 (murine embryonal carcinoma ATCC CRL 1720), P19 (murine embryonal carcinoma ATCC CRL 1825), SaOs2 (human osteogenic sarcoma ATCC HTB 85), H1299 (human lung adenocarcinoma cells obtained from M. Murphy), MDA231 (human breast adenocarcinoma ATCC HTB 26), MDA435 (human breast carcinoma ATCC HTB 129), T-47D (human breast carcinoma ATCC HTB 133), 293 (transformed human embryonic kidney cells ATCC CRL 1573), and SW480 (human colon adenocarcinoma ATCC CCL 228). Isolation of HERV-K LTRs. Primers specific for HERV-K LTRs were designed according to the published HERV-K10 LTR sequence (32): N5LTR1Kpn (5⬘-CGGGGTACCTGTTACTGTGTCTGTGTAG-3⬘; nucleotides [nt] 28 to 46) and N5LTR2Xho (5⬘-CCGCTCGAGTACACACCTGTGGGTGTT-3⬘; nt 949 to 932). The human teratocarcinoma cell line Tera 1 was used for PCR amplification of LTRs, most of which would presumably come from the more abundant solitary LTRs. The resulting PCR products were cloned upstream (Kpn-Xho sites) of a luciferase vector that includes a simian virus 40 (SV40) early promoter, PGL2-Promoter (Promega). To isolate LTRs from full-length proviral elements, a WI38 human fibroblast genomic library in the lambda FIXII vector (Stratagene) was screened with using an HERV-K gag probe (nt 1559 to 2037 from the HERV-K10 sequence). Lambda DNA from 6 of 10 isolated clones generated a PCR product with LTR-specific primers. Some of the LTRs were also cloned upstream of a LacZ vector devoid of any promoter (Promega parent pGEMZ vector with deleted promoter region). Luciferase and lacZ reporter gene assays. The appropriate reporter plasmids (1 to 2 ␮g) were transfected into various cell lines by either electroporation (Bio-Rad gene pulser with 0.4-cm cuvettes and 0.24 V) or LipofectAMINE (Gibco BRL) as specified by the supplier. For transient transfections, cells lysates were isolated 24 h after transfection and assayed for luciferase and lacZ light units by using the Dual Light system (Tropix) on a Perkin-Elmer microplate

9977

luminometer as specified by the manufacturer. Relative light unit levels were normalized for transfection efficiency with the converse reporter gene (Promega Luciferase PGL2-Control for lacZ; and Clontech LacZ pUC19-SV40 for luciferase). Pooled stable transfectants were established by cotransfecting a neo plasmid with the reporter gene of interest at a 1:10 molar ratio (pCI-neo mammalian expression vector; Promega) and selecting with G418 (Geneticin; Gibco BRL) for 1 week or until control cells with no neo plasmids died. X-Gal staining. Tissue culture cells were rinsed with phosphate-buffered saline (PBS) and fixed (2% formaldehyde, 0.2% gluteraldehyde, 1⫻ PBS) for 5 min at 4°C. The cells were washed again with PBS and overlaid with the stainingreaction mixture (1 mg of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside [XGal] per ml, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl2 in PBS). The cells were then incubated in a bacterial incubator with the reaction mixture for 12 to 18 h (the color can be seen in a few hours). Transgenic mice. The most active isolated LTR, LTRE3, was cloned upstream of the lacZ gene in a promoterless pGEM vector, and the LTRE3-lacZ sequences (devoid of vector sequences) were microinjected into fertilized eggs resulting from matings between B6D2F1 mice (C57BL/6J and DBA/2J hybrids) (16). Transgenic mice were identified by Southern blotting with 10 ␮g of restriction enzyme-digested tail DNA. The blots were hybridized with HERV-K LTR and ␤-galactosidase (␤-gal) probes. The tail DNA was also used for PCR analysis with primers for the HERV-K LTR and ␤-gal sequences (data not shown). Four separate transgenic lines were identified (Tg2, Tg4, Tg7, and Tg9), and two of those lines (Tg7 and Tg9) were subsequently found to express the ␤-gal gene by Northern or RT-PCR analysis or both. The nontransgenic line, 129Sv (Jackson Laboratory), was used as a negative control, while a transgenic line expressing lacZ in a ubiquitous fashion, ROSA␤-geo26 (12), was used as a positive control. Northern blots. Total RNA was extracted from frozen mouse testis, brain, lung, kidney, thymus, heart, ovary, uterus, skeletal muscle, spleen, intestine, and liver by using TRIzol (Gibco BRL) or RNEasy (Qiagen) as specified by the supplier. Poly(A)⫹ mRNA was isolated with the oligo(dT) cellulose MessageMaker (Gibco BRL) as recommended by the manufacturer. Northern blot analyses were performed by standard methods with 10 to 20 ␮g of total RNA or 10 ␮g of poly(A)⫹ mRNA. Formaldehyde-agarose gels (1.2% formaldehyde) were electrophoresed overnight, stained with ethidium bromide to examine 18S/28S RNA integrity, and transferred with Turboblotters onto Nytran nylon membranes as specified by the manufacturer (Schleicher & Schuell). The UVcrossed-linked membranes were then hybridized overnight in Church buffer (0.5 M Na2 HPO4/NaH2 PO4, 7% sodium dodecyl sulfate [SDS], 1 mM EDTA, 1% bovine serum albumin) with labeled HERV-K LTR probe. Probes were labeled with Stratagene Prime-it kits and added to the hybridization buffer at 106 cpm/ml at 65°C. The Northern blots were rinsed with 1⫻ SSC (0.15 M NaCl, 0.015 M sodium citrate)–0.1% SDS, washed with the same solution once at room temperature for 30 min, and washed with 0.1⫻ SSC–0.1% SDS twice at 65°C for 30 min. RT-PCR. One microgram of total RNA was treated with DNase as specified by the manufacturer (Gibco BRL) and reverse transcribed with the GeneAmp RNA PCR kit with oligo(dT) primers as specified by the vendor (Perkin Elmer). RT cycles consisted of 10 min at room temperature, 15 min at 42°C, 5 min at 99°C, and 5 min at 4°C with dT primers. The cDNAs were then subjected to 35 rounds of PCR amplification with primers (5⬘-GGGATGGCGTGGGACGCGGC-3⬘ and 5⬘-GGGACTGGGTGGATCAGTCGC-3⬘) specific for ␤-gal (the initial denaturation cycle was 94°C for 4 min; the amplification cycles were 45 s at 94°C, 45 s at 57°C, and 1 min at 72°C; and the extension cycle was 10 min at 72°C). RT-PCR controls included no reverse transcriptase added to the RT reaction mixture for one set of samples, to ensure that no contaminating DNA was present, and a different set of primers for the PCR (a gene expressed in most tissues, tpi), to assess the integrity of the starting RNA (5⬘-CACAAACACAAT ACAGGGGCTTTGGCACCGTG-3⬘ and 5⬘-GAACTGCTACAAAGTGACCA ATGGGCCTTTC-3⬘). Southern blots. Some of the RT-PCR products as well as tail DNA were run on agarose gels by Tris-acetate-EDTA gel electrophoresis. The DNA was denatured or neutralized in the gels, which were then transferred onto nylon membranes overnight by standard methods (Turboblotter and Nytran membranes). The labeled LTR probe (the same as in the Northern blot analyses) was added to the hybridization solution (5% Denhardt’s solution, 0.5% SDS, 6⫻ SSC) with the UV-cross-linked membrane for overnight hybridizations at 65°C. The Southern blots were rinsed and washed three times with 1⫻ SSC–0.1% SDS for 30 min each at 65°C. Additional washes with 0.1⫻ SSC–0.1% SDS were done when necessary. Immunofluorescence. Testis tissues were fixed in 4% paraformaldehyde (in PBS) overnight and then passed through a sucrose gradient (12, 16, and 18% sucrose solutions for 3 h each at 4°C). The tissues were embedded in tissue freezing medium (Triangle Biomedical Sciences) and flash frozen in liquid nitrogen. They were then sectioned in a cryostat at a thickness of 10 ␮m and used for immunofluorescence with a polyclonal ␤-gal antibody (Cortex Biochem). The sections were blocked with 10% goat serum in PBSBT (PBS, 0.5% bovine serum albumin, 0.05% Tween 20) for 30 min at room temperature. The polyclonal ␤-gal antibody was added (1:250), and the slides were incubated for 45 min at room temperature. The slides were washed three times for 5 min in PBSBT. A secondary antibody directly conjugated to Texas Red (1:500) was then added (goat anti-rabbit immunoglobulin G [Jackson ImmunoResearch]), and the slides were

9978

CASAU ET AL.

J. VIROL.

FIG. 1. Nucleotide sequence homology between HERV-K LTRE3 (KE3: nt 1 to 921) and HERV-K10 5⬘ LTR (K10: nt 78 to 999). Identities are depicted as dots. The TATA box and polyadenylation signal are capitalized. The potential glucocorticoid/enhancer site is underlined.

incubated for 30 min at room temperature. The sections were again washed three times for 5 min with PBSBT. Mounting solution (30% glycerol, 70% PBS) was used to mount the slides with coverslips. Nucleotide sequence accession numbers. The sequence determined in this study have been deposited in GenBank as follows: HERV-K LTRE3, AF148679.

RESULTS Isolation of active HERV-K LTRs. To isolate HERV-K LTRs from human genomic DNA, primers were designed from the published HERV-K10 LTR sequences (34). LTR PCR products from a teratocarcinoma cell line (Tera 1) were cloned upstream of a luciferase reporter gene vector (PGL2-Promoter), which includes its own SV40 early promoter, to identify LTRs harboring functional enhancers (data not shown). It had been previously shown (by investigating endogenous viral transcripts by Northern and RT-PCR analyses) that HERV-K family members can transcribe their viral genes as well as some adjacent cellular genes to high levels in testicular teratocarcinoma cell lines (GCT cell lines) but only to lower or nondetectable levels in most other tumors and cell lines tested to date (23, 25, 26, 45). However, no detailed studies investigating the expression patterns with isolated active LTRs have been done. To assess the general activity of the isolated LTRs, the cloned LTRs, driving the expression of the luciferase reporter gene were transiently transfected into GCT cell lines, Tera 1 and Tera 2, and into non-GCT cell lines, 293 (transformed embryonic kidney cell line) and H1299 (lung adenocarcinoma). Of 33 different LTR clones isolated by PCR selection, 18 were inactive in both GCT and non-GCT cell lines while 13 clones enhanced luciferase levels only a few-fold over promoter alone in GCT cell lines (data not shown). Two LTR clones were found to be highly active in GCT cell lines and not in non-GCT cell lines. The most active clone (LTRE3), with 10- to 100-fold activation in various GCT cell lines, was chosen for further study. The sequence of LTRE3 compared to that of HERVK10 LTR5⬘ is shown in Fig. 1. Both LTRs have a consensus TATA box and a polyadenylation signal, as well as a putative glucocorticoid/enhancer site. LTRE3 was shown to respond to the synthetic glucocorticoid hormone dexamethasone in GCT cells (fourfold activation upon addition of 10 mM dexamethasone) (data not shown).

Since the LTR PCR assay would probably favor selection of the 10,000 solitary LTRs rather than the 30 to 50 full-length proviruses, a human genomic library was screened with an HERV-K gag probe to isolate LTRs associated with 1 of the 50 or so full-length HERV-K proviruses in the genome. Of the 10 isolated lambda clones, 6 yielded PCR products with LTR specific primers. These LTRs were cloned into the luciferase reporter vector used previously and transfected in the various cell lines described above. Two of the six LTRs behaved similarly to the previously isolated LTRE3 (60 to 72% of LTRE3 activation), whereas the other four seemed to be relatively inactive in the in vitro assay described above (4 to 17% of LTRE3 activation) (data not shown). HERV-K LTR expression is restricted to teratocarcinoma cell lines. It has been previously observed that HERV-K expression (as determined by mRNA and protein expression as well as particle formation) is highest in testicular teratocarcinomas as well as in the cell lines derived from these tumors and is either undetectable or present at lower levels in a number of normal tissues and tumor types. Results with the isolated LTRE3 corroborate this conclusion. The enhancer(s) in LTRE3 increased luciferase expression relative to promoter alone from 10- to 70-fold in three human GCT cell lines (Tera 1, Tera 2, and 1218E) and in two murine GCT cell lines (F9 and P19) (Fig. 2A), but it did not enhance luciferase expression by more than 2-fold in several non-GCT human cell lines including a transformed embryonic kidney cell line, an osteogenic sarcoma, a lung adenocarcinoma, three breast carcinomas, and a colon adenocarcinoma (293, SaOs2, H1299, MDA231, MDA435, T47D, and SW480, respectively) (Fig. 2B). RA differentiation of a teratocarcinoma cell line leads to a decrease in LTR expression. Previous observations (8) have suggested the possibility that HERV-K LTR expression is dependent on the state of differentiation of GCTs. EC cells are the undifferentiated, rapidly dividing stem cell population of teratocarcinomas. The murine EC cell line F9 has been successfully used as an in vitro model to study the regulation of differentiation. These EC cells do not differentiate spontaneously but can be induced to do so upon treatment with the acidic form of vitamin A, retinoic acid (RA) (41). These EC cells can differentiate into primitive endoderm cells (when

VOL. 73, 1999


9979

FIG. 2. LTRE3 expression of a reporter gene is high in GCT cell lines (A) and low in non-GCT cell lines (B). The HERV-K LTRE3 was cloned upstream of the luciferase reporter gene and was transiently transfected into various cell lines along with a ␤-gal control vector to normalize for transfection efficiency. The relative luciferase level value is the light unit ratio between the luciferase vector (Pro-Luc) with promoter alone (SV40 early promoter) and the luciferase vector with promoter and LTRE3 (LTR-Pro-Luc).

treated with RA), into visceral endoderm (upon aggregation and treatment with RA), and into parietal endoderm (with RA and dibutyryl cyclic AMP) (41). This in vitro system was used to determine whether HERV-K expression is affected by the degree of differentiation in a GCT cell line. LTRE3 was cloned upstream of a bacterial lacZ expression plasmid which is devoid of any promoter sequences to test whether the LTR was able to act as its own promoter, using its intact TATA box and other regulatory elements (Fig. 1). F9 cells were induced to differentiate by adding 10⫺6 M RA to the medium for 1 week. Cells treated in this manner exhibited the characteristic morphological changes associated with differentiation (see Fig. 3B, panel 4). F9 cells and RA-differentiated F9 cells were transiently transfected either with a ␤-gal expression vector devoid of a promoter (lacZ vector) or one driven by LTRE3 (LTRE3-lacZ vector). A constitutively expressed luciferase vector (PGL2control vector) was also cotransfected to determine the efficiency of transfection in each cell population and normalize the results. LTRE3-driven lacZ expression in undifferentiated F9 cells (LTRE3-lacZ vector) was 30-fold higher than that of the basal levels of lacZ activity (lacZ vector) (Fig. 3A). However, LTRE3-driven lacZ expression in RA-differentiated F9 cells (LTRE3-lacZ vector) was reduced to fivefold compared to expression of the lacZ gene without the LTRE3 (Fig. 3A). Furthermore, LTRE3 expression was very low (less than twofold higher than basal levels) in the fully differentiated murine embryonic fibroblast cell line BALB/c 3T3 (data not shown). Additionally, X-Gal staining of pooled F9 clones stably transfected with LTRE3-lacZ and a neo vector (used to select for stably transfected cells), before and after RA differentiation, revealed a strong staining before differentiation and a lack of staining after differentiation (Fig. 3B). These observations demonstrate that HERV-K LTRE3 expression is sensitive to the degree of cell differentiation and hence may be developmentally regulated. Generation of transgenic mice harboring the LTRE3-lacZ sequence. Since LTRE3 was shown to be active in murine cells

(Fig. 3), it was postulated that this human LTR could be tested for tissue specificity in its ability to drive transcription in a transgenic mouse system. The same construct used in the RA differentiation experiment in Fig. 3 was used to create transgenic mice harboring the human HERV-K LTRE3 driving the expression of the bacterial lacZ gene. Four mice were shown to be transgenic for the human endogenous viral LTR and the bacterial lacZ gene by both Southern blot and PCR analyses of tail DNA (data not shown). The four independent transgenic lines (Tg2, Tg4, Tg7, and Tg9) were then crossed to 129Sv mice to generate mouse lines and homozygotes of all four independent transgenic lines. Transgene expression is restricted mainly to the more undifferentiated spermatocytes of adult testes. Total RNA was extracted from mouse tissues (brain, liver, lung, thymus, heart, intestines, testis, ovaries, uterus, skeletal muscle, kidney, and spleen) and used for RT-PCR as well as Northern blot analysis. Of the four transgenic lines obtained, two (Tg7 and Tg9) were shown to express the lacZ gene in a few tissues and to various degrees. Tg7 and Tg9 were both found to express lacZ in adult testes when analyzed by RT-PCR (Fig. 4A). The control experiment with no reverse transcriptase enzyme (⫺RT) showed no bands even when the RT-PCR gel was assayed by Southern blotting and long exposures were taken. This demonstrates that there is no DNA contamination in the RT-PCR reactions. The ROSA␤-geo26 strain (R⫹) was used as a positive control since it constitutively expresses lacZ in most of its organs and tissues. The 129Sv strain (S⫺) was used as a negative control since it does not contain any lacZ sequences. Northern blot analysis of total RNA from the two expressing transgenic lines revealed that Tg7 expresses discrete sized transcripts in adult testes while Tg9 does not (expression was detectable only by RT-PCR analysis), reflecting differences in the levels of expression between the two independently derived transgenic lines. The lacZ transgenic line ROSA␤-geo26 and the nontransgenic line 129Sv were used to control for the specificity of the lacZ probe (Fig. 4A and C). mRNA was also isolated from both

9980

CASAU ET AL.

J. VIROL.

FIG. 3. LTRE3 expression is downregulated upon differentiation of an EC cell line. (A) The HERV-K LTRE3 was cloned upstream of a lacZ reporter gene in a vector devoid of any promoter sequences and transiently transfected into the murine EC cell line, F9, along with a luciferase control vector to normalize for transfection efficiency. The relative lacZ level value is the ratio of the promoterless lacZ vector with that of the other vectors: no lacZ (no DNA as a negative control), LTR-lacZ (LTRE3-lacZ), and SV40 lacZ (lacZ vector with the SV40 promoter as a positive control). (B) F9 cells were also stably transfected with the LTRE3-lacZ vector and a neo vector to select for G418-resistant colonies. Colonies were pooled and stained with X-Gal to look for lacZ staining. Panel 1 shows the F9 cells stained with X-Gal. Panel 3 shows the same field by phase-contrast microscopy to show cell morphology. This pooled stable F9 cell line was also differentiated for 1 week with 10⫺6M RA. Panel 2 shows the differentiated F9 cells stained with X-Gal. Panel 4 shows the same field by phase-contrast microscopy to show the differentiated cell morphologies.

transgenic lines but, as with Northern blot analysis with total RNA, no detectable expression was seen for Tg9 (Fig. 4D). RT-PCR analysis of other mouse tissues revealed that lacZ expression driven by the HERV-K LTR is indeed limited primarily to the testes (Fig. 5). RT-PCR analysis was carried out in multiple experiments, each done in triplicate to minimize the nonquantitative nature of PCR amplifications. In addition to the positive signals in the testes, only brain tissues generated positive signals by RT-PCR (consistently high in Tg9 and lower in Tg7). Tissues such as kidney, liver, and thymus at times produced faint bands, but these RT-PCR signals were not reproducibly consistent. The other tissues did not generate reproducible signals by RT-PCR (Fig. 5) and did not generate signals by Northern blot analysis (data not shown).

To determine the cell types in the seminiferous tubules of adult testis that are responsible for HERV-K LTR expression, frozen testis sections were used for immunofluorescence studies with a ␤-gal polyclonal antibody specific for the bacterial LacZ protein product. The strongest staining was observed in the testes of the positive control ROSA␤-geo26 mice (ROSA⫹) (Fig. 6A). This staining was restricted primarily to the more differentiated cell types of the seminiferous tubules facing the interior of the lumen. As negative controls, testes sections from the ROSA⫹ line were also stained with primary antibody only (anti-␤-gal) (Fig. 6B) and secondary antibody only (Texas Red) (Fig. 6C). Compared to the nontransgenic 129Sv sections (Fig. 6D to F), in which faint background staining was visible, sections of testes from Tg7 mice (Fig. 6G to I)

FIG. 4. Two independent transgenic lines express the lacZ gene in adult testes, but at different levels. (A) RT-PCR analysis followed by Southern blotting reveals that only lines Tg7 and Tg9 express the reporter gene. R⫹ refers to the ROSA␤-geo26 lacZ line, S⫺ refers to the nontransgenic control line, while Tg2, Tg4, Tg7, and Tg9 refer to the transgenic lines. Testis RNA was DNase treated and reverse transcribed in the ⫹RT gel (but not in the ⫺RT gel) and PCR amplified with lacZ primers. (B) RNA integrity was checked by using the RT reactions to PCR amplify a ubiquitous gene, tpi. (C) Total testis RNA was used for Northern blot analysis and hybridized with a lacZ probe. (D) mRNA was isolated for Tg7 and Tg9 and hybridized to a lacZ probe.

VOL. 73, 1999


9981

FIG. 5. RT-PCR analysis of tissue RNA from transgenic mice reveals the strongest expression in adult testes and, to a lesser extent, in adult brain. Tissues selected from the two expressing transgenic lines Tg7 and Tg9 were Br (brain), Ht (heart), Ts (testis), It (intestines), Kd (kidney), Lg (lung), Lv (liver), Sk (skeletal muscle), Sp (spleen), Th (thymus), Ut (uterus), and Ov (ovary). Controls included negative control tissues from 129Sv nontransgenic mice (S⫺) and positive control tissues from a constitutive lacZ-expressing ROSA␤-geo26 strain (R⫹). All isolated RNAs were DNase treated for 30 min, and 1 ␮g was used for each RT-PCR. The first row (⫹RT ␤-gal) is RNA that was reverse transcribed with dT primers and PCR amplified with ␤-gal primers. The second row (⫺RT ␤-gal) is RNA that was not reverse transcribed but was PCR amplified with ␤-gal primers. The third row (⫹RT Tpi) is the same reverse-transcribed RNA as in the first row but PCR amplified with tpi primers (a ubiquitously expressed gene).

exhibited a more intense staining, which appeared to be restricted primarily to the more undifferentiated spermatocytes in the seminiferous tubules. DISCUSSION This family of viruses is thought to be the most active HERV group in the human genome due to its high levels of expression in some tumor cell lines and its ability to code for functional proteins that can give rise to particles, albeit immature noninfectious ones. These proviruses have been maintained in the human genome for more than 30 million years. Although these proviruses may be regarded as vestiges of the past, they remain active, with the potential to impact differentiated or pathological states. It remains possible that the expression of at least one of the HERV-K viral genes or some cellular genes driven by the retroviral LTRs has been selected for and that this is the reason why some have not been mutated out of existence. It was previously observed that HERV-K proviruses preferentially express their viral genes in GCTs such as testicular teratocarcinomas. HERV-K expression has also been found in some other tissue and tumor types, but it has been sporadic and has been present at much lower levels. No thorough studies had been undertaken to determine the expression patterns dictated by isolated active HERV-K LTRs. To address how this family of virus regulates the expression of its viral genes or neighboring cellular genes, it was necessary to isolate and characterize an active LTR. The results in this paper demonstrate that an isolated active

HERV-K LTR confers the same types of expression patterns that had been observed for the viral genes. An active HERV-K LTRE3 was able to drive the expression of reporter genes in testicular teratocarcinoma cell lines but not in a variety of other cell lines including a transformed kidney cell line, breast carcinomas, an osteosarcoma, and lung and colon adenocarcinomas. Furthermore, this LTR was downregulated upon differentiation of a teratocarcinoma cell line, indicating that it might be developmentally regulated. A transgenic mouse model was used to determine that expression in the adult tissues was limited to the testes and, to a lesser extent, the brain. Although the expression of some HERV-K elements was previously noted in the placenta (39), no expression was found in the placenta of near-term LTRE3 transgenic pups by RT-PCR (data not shown). The strong possibility remains that there are some HERV-K LTRs which generate other expression patterns (such as placenta expression or differentiated trophectoderm expression) due to sequence variations that are still uncharacterized. All expression patterns were noted in two independent transgenic lines and were stable over all generations tested. A few other tissues did, in some RT-PCR experiments, generate faint signals, but the irregularity of these signals and their low intensities suggest that they might be considered low basal levels of expression. In contrast, RT-PCR-detected expression in testis was very reproducible and always generated strong signals in Tg7, and the expression was also seen by Northern blot analysis. Tg9 expressed the reporter gene to lower levels in

9982

CASAU ET AL.

FIG. 6. Immunofluorescence studies reveal staining in the more undifferentiated spermatocytes of the testes of transgenic line Tg7. Frozen sections of adult testes were incubated with a polyclonal antibody against ␤-gal to detect lacZ staining. (A) ROSA⫹ control with primary ␤-gal antibody and secondary Texas Red antibody. (B) ROSA⫹ with only primary ␤-gal antibody. (C) ROSA⫹ with only secondary Texas Red antibody. (D to F) Three sections of nontransgenic testes stained with ␤-gal and Texas Red antibodies. (G to I) Three sections of transgenic line Tg7 stained with ␤-gal and Texas Red antibodies. ROSA⫹ shows strongest staining in the more differentiated cell types of the seminiferous tubules (found near the lumen of the seminiferous tubules), while the negative control 129Sv has a faint amount of background. Sections of testes from Tg7 exhibit staining which is restricted primarily to the more undifferentiated spermatocytes found on the periphery of the seminiferous tubules.

J. VIROL.

preferentially expressed in germ cells, as noted by its high levels of expression in undifferentiated spermatocytes and testicular teratocarcinomas. Since LTRs drive gene expression by interacting with a variety of ubiquitous and cell-type-specific transcription factors, additional HERV-K LTR mutational and deletion studies to identify proteins that are involved in the regulation of proliferation and differentiation of germ cells and other stem cells are under way. Testicular teratocarcinomas are the primary malignancy that have long been associated with high levels of HERV-K expression as well as particle formation. However, the significance of these findings with respect to HERV-K gene expression (i.e., whether it is involved in or merely a consequence of the development of these tumors) remains unclear. The data presented in this paper support the previous observations that HERV-K proviruses are expressed primarily in GCTs containing EC components. We contributes the novel finding that the differentiation of a GCT leads to a dramatic decrease in HERV-K LTR expression. Furthermore, the LTR is also found to be preferentially expressed in the more undifferentiated spermatocytes of adult testes. Taken together, it can now be concluded that active HERV-K members can be expressed not only in GCTs but also in healthy adult germ cells. Experiments to cross the LTRE3 transgenic mice with strains of mice that have a high predisposition to testicular cancer are under way. This should be useful in determining the extent to which this virus is expressed as the cancer develops. ACKNOWLEDGMENTS We thank A. Teresky for help in the initial setup of the mouse colonies. We are indebted to J. Moran, B. Prabhakaran, P. Robinson, J. Levorse, M. Shin, and C. Unrine for helpful suggestions and protocols for the analysis of the transgenic mice. We also thank A. Muller for helpful discussions and critical reading of the manuscript. This work was supported by cancer training grant 5T32 CA09528-14 from the National Institutes of Health. REFERENCES

testes and to slightly higher ones in brain than did Tg7. The significance of the expression in the brain is unclear, but it is worthwhile to note that a transgenic mouse harboring a murine endogenous retrotransposon LTR (IAP) directing lacZ expression was also found to only express the reporter gene in testes and brain (10). IAPs are murine proviral elements that produce particles which assemble on the membranes of the endoplasmic reticulum and subsequently bud into the cisternae (19). These particles, like the HERV-K HTDVs, are noninfectious and cannot seemingly be transmitted horizontally. As with HERV-K particles, IAPs seem to be present in many testicular teratocarcinomas with an EC component but absent or present in fewer numbers in more highly differentiated derivatives of these GCTs (19, 43). HERV-K and IAP elements are not evolutionarily related and yet are expressed in very similar patterns. Furthermore, many other HERVs, such as HERV-H and ERV-9 family members, are likewise expressed primarily in GCTs (25). The significance of these independently derived similar expression patterns is unclear, although germ line expression can ensure their survival. Since endogenous retroviruses such as HERV-K are transmitted vertically and do not seemingly have an exogenous phase where they would be able to infect other cells and other hosts, it is critical for their amplification or movement in the genome that they be expressed in germ cells, where any reintegration events can be passed on to the next generation. It is therefore not surprising that HERV-K does indeed seem to be

1. Andrews, P. W. 1988. Human teratocarcinomas. Biochim. Biophys. Acta 948:17–36. 2. Andrews, P. W., and I. Damjanov. 1994. In R. J. Hay, J. Park, and A. Gazder (ed.), Atlas of human tumor cell lines, p. 443–476. Academic Press, Inc., New York, N.Y. 3. Baltimore, D. 1985. Retroviruses and retrotransposons: the role of reverse transcription in shaping the eukaryotic genome. Cell 40:481–482. 4. Boller, K., H. Frank, J. Lower, R. Lower, and R. Kurth. 1983. Structural organization of unique retrovirus-like particles budding from human teratocarcinoma cell lines. J. Gen. Virol. 64:2549–2559. 5. Brack-Werner, R., C. Leib-Mosch, T. Werner, V. Erfle, and R. Hehlmann. 1989. Human endogenous retrovirus-like sequences. Hamatol. Bluttransfus. 32:467–477. 6. Brodsky, I., B. Foley, and D. Gillespie. 1993. Expression of human endogenous retrovirus (HERV-K) in chronic myeloid leukemia. Leuk. Lymphoma 11:119–123. 7. Brodsky, I., B. Foley, D. Haines, J. Johnston, K. Cuddy, and D. Gillespie. 1993. Expression of HERV-K proviruses in human leukocytes. Blood 81: 2369–2374. 8. Bronson, D. L., W. C. Saxinger, D. M. Ritzi, and E. E. Fraley. 1984. Production of virions with retrovirus morphology by human embryonal carcinoma cell in vitro. J. Gen. Virol. 65:1043–1051. 9. Coffin, J. 1984. RNA tumor viruses, vol. 1, p. 1109–1203. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 10. Dupressoir, A., and T. Heidmann. 1996. Germ line-specific expression of intracisternal A-particle retrotransposons in transgenic mice. Mol. Cell. Biol. 16:4495–4503. 11. Feuchter-Murthy, A., J. Freeman, and D. Mager. 1993. Splicing of a human endogenous retrovirus to a novel phospholipase A2 related gene. Nucleic Acids Res. 21:135. 12. Friedrich, G., and P. Soriano. 1991. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5:1513–1523. 13. Harris, J. M., R. H. Haynes, and E. M. McIntosh. 1997. A consensus sequence for a functional human endogenous retrovirus K (HERV-K)

VOL. 73, 1999 dUTPase. Biochem. Cell. Biol. 75:143–151. 14. Herbst, H., M. Sauter, C. Kuhler-Obbarius, T. Loning, and N. MuellerLantzsch. 1998. Human endogenous retrovirus (HERV)-K transcripts in germ cell and trophoblastic tumours. APMIS 106:216–220. 15. Herbst, H., M. Sauter, and N. Mueller-Lantzsch. 1996. Expression of human endogenous retrovirus K elements in germ cell and trophoblastic tumors. Am. J. Pathol. 149:1727–1735. 16. Hogan, B., F. Constantini, and E. Lacy. 1986. Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Kitamura, Y., T. Ayukawa, T. Ishikawa, T. Kanda, and K. Yoshiike. 1996. Human endogenous retrovirus K10 encodes a functional integrase. J. Virol. 70:3302–3306. 18. Krieg, A. M., M. F. Gourley, and A. Perl. 1992. Endogenous retrovirus: potential etiologic agents in autoimmunity. FASEB J. 6:2537–2544. 19. Kuff, E. L., and K. K. Lueders. 1988. The intracisternal A-particle gene family: structure and functional aspects. Adv. Cancer Res. 51:183–276. 20. Larsson, E., A. Anderson, L. Holmberg, R. Ohlsson, N. Kato, J. Callacio, and M. Cohen. 1993. Expression of an endogenous retrovirus, HERV-R, in human tissues. J. Cancer Res. Clin. Oncol. 119:S6. 21. Leib-Mosch, C., M. Haltmeier, T. Werner, E. Geigl, R. Brack-Werner, U. Francke, V. Erfle, and R. Hehlmann. 1993. Genomic distribution and transcription of solitary HERV-K LTRs. Genomics 18:261–269. 22. Lower, J., R. Lower, J. Stegmann, H. Frank, and R. Kurth. 1981. Retrovirus particle production in three of four human teratocarcinoma cell lines, p. 541–544. In R. Neth, R. C. Gallo, T. Graf, and K. Mannweiler (ed.), Modern trends in human leukemia, vol. IV. Springer-Verlag, Berlin, Germany. 23. Lower, R., K. Boller, B. Hasenmaier, C. Korbmacher, N. Muller-Lantzsch, J. Lower, and R. Kurth. 1993. Identification of human endogenous retroviruses with complex mRNA expression and particle formation. Proc. Natl. Acad. Sci. USA 90:4480–4484. 24. Lower, R., J. Lower, H. Frank, R. Harzmann, and R. Kurth. 1984. Human teratocarcinomas cultured in vitro produce unique retrovirus-like viruses. J. Gen. Virol. 65:887–898. 25. Lower, R., J. Lower, and R. Kurth. 1996. The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc. Natl. Acad. Sci. USA 93:5177–5184. 26. Lower, R., J. Lower, C. Tondera-Koch, and R. Kurth. 1993. A General method for the identification of transcribed retrovirus sequence (R-U5PCR) reveals the expression of the human endogenous retrovirus loci HERV-H and HERV-K in teratocarcinoma cells. Virology 192:501–511. 27. Mayer, J., E. Meese, and N. Mueller-Lantzsch. 1997. Chromosomal assignment of human endogenous retrovirus K (HERV-K) env open reading frames. Cytogenet. Cell Genet. 79:157–161. 28. Mayer, J., E. Meese, and N. Mueller-Lantzsch. 1997. Multiple human endogenous retrovirus (HERV-K) loci with gag open reading frames in the human genome. Cytogenet. Cell Genet. 78:1–5. 29. Mayer, J., M. Sauter, A. Racz, D. Scherer, N. Mueller-Lantzsch, and E. Meese. 1999. An almost-intact human endogenous retrovirus K on human chromosome 7. Nat. Genet. 21:257–258. 30. Medstrand, P., and D. L. Mager. 1998. Human specific integrations of the HERV-K endogenous retrovirus family. J. Virol. 72:9782–9787. 31. Miki, Y., I. Nishisho, A. Horii, Y. Miyoshi, J. Utsunomiya, K. Kinzler, B. Vogelstein, and Y. Nakamura. 1992. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res. 52:643–645. 32. Morse, B., P. Rotherg, V. South, J. Spandorfer, and S. Astrin. 1988. Inser-


33.

34. 35. 36.

37. 38. 39.

40.

41. 42.

43. 44. 45.

46. 47. 48.

9983

tional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature 333:87–90. Mueller-Lantzsch, N., M. Sauter, A. Weiskircher, K. Kramer, B. Best, M. Buck, and F. Grasser. 1993. Human endogenous retroviral element K10 (HERV-K10) encodes a full-length Gag homologous 73-kDa protein and a functional protease. AIDS Res. Hum. Retroviruses 9:343–350. Ono, M. 1986. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to type A and B retrovirus genes. J. Virol. 58:937–944. Ono, M., T. Yasunaga, T. Miyata, and H. Ushikubo. 1986. Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. J. Virol. 60:589–598. Rasmussen, H. B., C. Geny, L. Deforges, H. Perron, W. Tourtelotte, A. Heltberg, and J. Claussen. 1995. Expression of endogenous retroviruses in blood mononuclear cells and brain tissue from multiple sclerosis patients. Multiple Sclerosis 1:82–87. Rasmussen, H. B., H. Perron, and J. Clausen. 1993. Do endogenous retroviruses have etiological implications in inflammatory and degenerative nervous diseases? Acta Neurol. Scand. 88:190–198. Simon, M., M. Haltmeier, G. Papakonstantinou, T. Werner, R. Hehlmann, and C. Leib-Mosch. 1994. Transcription of HERV-K-related LTRs in human placenta and leukemic cells. Leukemia 1994(Suppl. 1):S12–S17. Simpson, G. R., C. Patience, R. Lower, R. R. Tonjes, H. D. M. Moore, R. A. Weiss, and M. T. Boyd. 1996. Endogenous D-type (HERV-K) related sequences are packaged into retroviral particles in the placenta and possess open reading frames for reverse transcriptase. Virology 222:451–456. Steinhuber, S., M. Brack, G. Hunsmann, H. Schwelberger, M. Dierich, and W. Vogestseder. 1995. Distribution of human endogenous retrovirus HERV-K genomes in humans and different primates. Hum. Genet. 96:188– 192. Strickland, S., and V. Mahdavi. 1978. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393–403. Tassabehji, M., T. Strachan, M. Anderson, D. R. Campbell, S. Collier, and M. Lako. 1994. Identification of a novel family of human endogenous retroviruses and characterization of one family member, HERV-K(C4), located in the complement C4 gene cluster. Nucleic Acids Res. 22:5211–5217. Teresky, A. K., M. Marsden, E. L. Kuff, and A. J. Levine. 1974. Morphological criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice. J. Cell. Physiol. 84:319–332. Tonjes, R. R., K. Boller, C. Limbach, R. Lugert, and R. Kurth. 1997. Characterization of human endogenous retrovirus K virus-like particles generated from recombinant baculoviruses. Virology 233:280–291. Tonjes, R. R., R. Lower, K. Boller, J. Denner, B. Hasenmaier, H. Kirsch, H. Konig, C. Korbmacher, C. Limbach, R. Lugert, R. C. Phelps, J. Scherer, K. Thelen, J. Lower, and R. Kurth. 1996. HERV-K: the biologically most active human endogenous retrovirus family. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 13(Suppl. 1):S261–S267. Urnovitz, H. B., and W. H. Murphy. 1996. Human endogenous retroviruses: nature, occurrence, and clinical implications in human disease. Clin. Microbiol. Rev. 9:72–99. Wilkinson, D. A., D. L. Mager, and J. C. Leong. 1994. Endogenous human retroviruses, p. 465–535. In J. A. Levy (ed.), The Retroviridae, vol. 3. Plenum Press, New York, N.Y. Zhu, Z., S. Hsieh, D. R. Bentley, D. R. Campbell, and J. E. Volanakis. 1992. A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus. J. Exp. Med. 175:1783–1787.