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Journal of Virology, November 1999, p. 9496-9507, Vol. 73, No. 11
Paul-Ehrlich-Institut, D-63225 Langen,
Germany
Received 14 June 1999/Accepted 6 August 1999
cORF, a protein encoded by the human endogenous retrovirus family
HTDV/HERV-K, contains amino acid motifs which resemble the nuclear
import and export signals of the viral regulatory proteins Rev (human
immunodeficiency virus) and Rex (human T-cell leukemia virus [HTLV]).
In this study, we demonstrated that cORF indeed has a Rev-like function
and mapped the cORF-responsive RNA element to a sequence in the 3' long
terminal repeat, a localization similar to RxRE, the responsive element
in HTLV type 1. Accordingly, we have given the element the designation
RcRE. cORF and RcRE stabilize unspliced and incompletely spliced viral
transcripts and enhance their nuclear export via the CRM1 export
pathway. So far, HTDV/HERV-K is the only endogenous retrovirus family
with a complex regulation at the posttranscriptional level. It may be
regarded as an intermediate in the evolution from simple to complex retroviruses.
All retroviruses have three genes in
common: gag, the protease/polymerase gene
(prt/pol), and the envelope gene (env). Complex retroviruses possess additional accessory genes, some of which regulate
the expression of viral genes at the transcriptional or the
posttranscriptional level. In the human immunodeficiency virus (HIV),
for example, inefficient splice sites and so-called instability
sequences in the viral introns cause nuclear retention or degradation
of primary transcripts (30, 34, 43) unless these transcripts
are completely spliced to small mRNAs encoding inter alia the
regulatory proteins Tat and Rev (6). Efficient nuclear
export of full-length transcripts and subgenomic env
transcripts depends on the interaction of two viral elements, the
regulator of expression of viral proteins (Rev) and a Rev-responsive
RNA element (RRE) (15, 42). The equivalent elements of human
T-cell leukemia virus (HTLV) are designated Rex and RxRE, respectively.
Transport of Rev and Rev homologues into the nucleus is mediated by the
interaction of a domain rich in basic amino acids, the nuclear
localization signal (NLS), with cellular import factors (18)
(for reviews, see references 6, 36, and
46). In the nucleus, the viral protein binds to its
responsive element in the primary viral transcript, forming multimers.
In cooperation with a plethora of cellular factors, the Rev-RRE or
Rex-RxRE interaction leads to suppression of splicing, to stabilization
of full-length and singly spliced transcripts in the nucleus, to
increased export of such transcripts to the cytoplasm, and thus
eventually to the generation of infectious progeny (reviewed, e.g., in
references 19 and 41). The
nuclear export signal of Rev and Rev homologues most often consists of
a motif enriched in leucine residues (11). CRM1, the nuclear
export factor for leucine-rich nuclear export signals (NESs), is the
most important of the cellular factors which have been found to bind to
this signal (12, 13, 35). CRM1 is specifically inhibited
upon binding to the antibiotic leptomycin B (LMB) (4, 16, 23, 39,
48).
The responsive RNA sequences to which proteins with Rev-like function
bind are located in quite different parts of the respective viral
transcripts. The Rev-responsive element of HIV (RRE) is located in
env (31), while the Rex-responsive element (RxRE) of HTLV type 1 (HTLV-1) is located in the 3' U3R (1). In
several test systems, Rex as well as Rev can cross-act with responsive elements of related viruses (reviewed in reference
10). In summary, the regulation of gene expression
by the interaction of a Rev-like protein with a responsive RNA element
is a hallmark of complex retroviruses.
Endogenous retroviruses (ERVs) are integral parts in the genomes of
many, if not of all, species. ERVs most probably resulted from
infection of germ line cells with exogenous retroviruses and subsequent
fixation of their genetic information in the host genome. While ERVs
related to simple retroviruses have been well known for many years, it
is intriguing that no endogenous counterparts of complex retroviruses
have yet been detected (28). In this report, we show
evidence that the human ERV (HERV) family HERV-K is an exception. This
is the only HERV family known to code for retroviral particles, the
human teratocarcinoma-derived virus (HTDV) particles described more
than a decade ago (5, 24-26). The family comprises 30 to 50 proviruses in the genomes of humans and of Old World monkeys
(38). Their closest contemporary relatives are mammalian
type B and D retroviruses. A prototypic HTDV/HERV-K genome (type 2)
(27) with its mRNA transcripts is depicted in Fig.
1A.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
cORF and RcRE, the Rev/Rex and RRE/RxRE Homologues of the Human
Endogenous Retrovirus Family HTDV/HERV-K
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
(A) Genomic organization and transcripts of HTDV/HERV-K
type 2. SA, splice acceptor; SD, splice donor. (B) Comparison of
arginine-rich NLSs. (C) Comparison of leucine-rich NESs. Amino acids in
boldface are essential for function.
Like all hitherto identified HERVs, HTDV/HERV-K proviruses are not infectious, although complete open reading frames for all viral genes have been observed in cDNA clones and recently also in a full-length provirus on chromosome 7 (32). Their potential for encoding functional proteins has been studied in particle-producing cell lines and with recombinant expression systems (28, 45). HTDV/HERV-K mRNA expression in teratocarcinoma cell lines resembles the pattern seen with complex retroviruses: full-length transcripts and env transcripts are accompanied by alternatively spliced small mRNA species (Fig. 1A) (26). One of these transcripts, a 1.8-kb doubly spliced mRNA, codes for a protein of 14 kDa designated cORF, which contains a putative arginine-rich NLS (Fig. 1B) and a putative leucine-rich NES (Fig. 1C) and which is targeted to the nucleoli similarly to Rev and Rex (27, 29).
This similarity to regulatory proteins of complex exogenous retroviruses prompted us to investigate whether efficient protein expression in the HTDV/HERV-K family also depends on Rev-RRE-like regulatory elements. In this study, we demonstrated that cORF indeed has a Rev/Rex-like function and mapped the responsive element. We showed that cORF stabilizes incompletely spliced mRNAs in the nucleus and enhances their transport to the cytoplasm when the cORF-responsive element is present. Furthermore, we present data indicating that cORF uses the CRM1 nuclear export pathway. HTDV/HERV-K is the first example of an ERV with similarities to complex retroviruses.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cells. COS7 (African green monkey kidney) and HeLa (human cervical carcinoma) cells were obtained from the American Type Culture Collection. The teratocarcinoma cell line GH had been established earlier (24). HLtat is a HeLa derived cell line stably transfected with a Tat-expressing plasmid. All cell lines were grown in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum (PAA Laboratories), 2 mM glutamine, and antibiotics. Cells were subdivided weekly as follows: COS7, 1:20; HeLa, 1:40; GH, 1:3; and HLtat, 1:30.
Plasmid constructions.
Two HIV expression plasmids were
kindly provided by B. Felber, Frederick, Md.: (i) plasmid pNLcgagA2, a
derivative of plasmid pCgagA2, here called pHIVgagRRE; and (ii) plasmid
pBsrev, here designated pREV. Two control plasmids were constructed as
follows: pHIVgag, by removing the RRE in pHIVgagRRE by the use of
restriction endonucleases SacII and XhoI,
treatment with the Klenow fragment of DNA polymerase to generate blunt
ends, and ligation; and p(
), by removing most of the Rev-coding
sequence in pBsrev by the use of HindIII and
EcoRI, filling in recessive ends with Klenow enzyme, and
blunt-end ligation. All enzymes were purchased from either New England
Biolabs or Gibco BRL.
cells
(Gibco BRL). For transfections, plasmids were prepared with an
endotoxin-free plasmid preparation kit (Qiagen).
Transfections. Transfections were performed either in six-well plates (Greiner) containing coverslips (for immunofluorescence studies), in 25-cm2 tissue culture flasks (Nunc) (for p24 quantitation), or in tissue culture dishes (diameter, 14.5 cm) (for RNA preparations). Cells were seeded at a density of 1 × 106 (six-well plates), 2 × 106 (flasks), or 2.5 × 106 (dishes) the day before transfection. On the day of transfection, the cells were 50 to 70% confluent (for RNA preparations they were less than 50% confluent). Transfections were carried out with DOTAP liposomal transfection reagent (Boehringer Mannheim) or Lipofectamine Plus (Gibco BRL) in accordance with the users' manual. The total amount of DNA used in transfections was 4 µg in six-well plates and 8 µg in bottles with DOTAP, respectively, or 1 µg in six-well plates, 1.5 µg in bottles, and 6 µg in dishes with Lipofectamine Plus. In cotransfections, vectors were used in equimolar amounts. Cells were incubated in serum-free transfection medium for 5 to 6 h at 37°C, after which the transfection medium was changed to medium containing 10% fetal calf serum. The cells were processed for further studies 24 to 48 h after transfection.
For transfections with LMB, 2 to 6 nM LMB was added at the time of transfection and cells were treated with constant amounts of LMB until the time of fixation or lysis.Immunocytochemistry.
Cells on coverslips were fixed 24 or
48 h posttransfection, and immunofluorescence staining was
performed as described previously (5). The following
antibodies and dilutions were used: anti-cORF (-cORF; rabbit), 1:200
to 1:500 (27); -HERV-K Gag (rabbit), 1:500 to 1:1,000
(26); -HERV-K Gag (goat), 1:500 to 1:1,000; -HERV-K
Env TM-env (goat), 1:500 to 1:1,000; and -HIV p24 monoclonal (mouse), 1:400 (Du Pont). The -HERV-K Gag (goat) antibody was provided by R. R. Tönjes, Langen, Germany; the -HERV-K
Env TM-env (goat) antibody was donated by J. Denner, Langen, Germany.
Secondary antibodies and their dilutions were as follows: fluorescein
isothiocyanate (FITC)-conjugated goat -rabbit immunoglobulin G
(IgG), 1:400 (Cappel); FITC-conjugated donkey -rabbit IgG, 1:25 to
1:50 (Amersham); Cy3-conjugated donkey -goat IgG, 1:500 to 1:1,000
(Dianova); and Cy3-conjugated goat -mouse IgG, 1:500 to 1:1,000
(Dianova). For double immunofluorescent staining, two primary as well
as two secondary antibodies were mixed and incubations were carried out
simultaneously. After final washes in phosphate-buffered saline (PBS),
cells were mounted in Moviol (Hoechst, Frankfurt) on slides. Preparations were examined by using a Zeiss Axiophot microscope equipped with a fluorescence illuminator with appropriate filters. Kodak Ektachrome 400 X color reversal film was used for documentation.
Quantitation of p24. Two days after transfection in the presence of DOTAP, Gag particles were prepared from cell culture supernatants of transfected cells and duplicates were combined. After centrifugation at 10,000 × g and 4°C for 15 min to remove cellular components, supernatants were centrifuged at 100,000 × g and 4°C for 80 min. The resulting pellet was dissolved in 200 µl of cell culture lysis buffer (Promega). Volumes of 200 µl of diluted cell lysate or entire particle preparations were employed in the HIV AG-1 Monoclonal p24 capture assay (Abbott). One day after transfection in the presence of Lipofectamine Plus or 2 days after transfection in the presence of DOTAP, cells were lysed with 500 µl of cell culture lysis buffer per bottle after two washes with PBS. The lysate was centrifuged for 30 s at 4°C and 10,000 rpm in a Biofuge R centrifuge (Heraeus). Total protein contents of cell lysates were determined by the use of the Bio-Rad protein assay (Bio-Rad) according to the users' manual. Depending on the expected concentration of HIV Gag, 1 to 5 µl of the cell lysate were diluted to 240 µl with 0.5× lysis buffer. The test was carried out according to the manufacturer's instructions.
Immunoblotting.
Cell lysates of the same transfectants as
for the p24 capture assay were used for immunoblotting. Proteins were
separated in 12.5 or 10% polyacrylamide gels under reducing
conditions. Fifteen microliters (DOTAP) or 2 µl (Lipofectamine Plus)
of lysate was applied per lane. The proteins were transferred to
polyvinylidene difluoride membranes (Hybond-P; Amersham). The membranes
were blocked with 5% nonfat dried milk (Marvel) (see Fig. 2) or 5% nonfat dried milk plus 2% bovine serum albumin fraction V (Boehringer Mannheim) (see Fig. 5) and 0.1% Tween 20 (Serva) in PBS. After being
washed with PBS containing 0.1% detergent, membranes were incubated
for 1 h at room temperature with an HIV-positive human serum
specimen at a dilution of 1:500. Membranes were washed extensively and
incubated with a second antibody conjugated with horseradish peroxidase: -human IgG (Sigma) at a dilution of 1:2,000 to 1:5,000. Immunoreactive proteins were detected by the use of an enhanced chemiluminescence kit (ECL+Plus; Amersham).
Northern blotting. Cytoplasmic and nuclear RNA was prepared with an RNeasy Mini Kit (Qiagen) in accordance with the manufacturer's instructions. mRNA preparation and Northern blotting conditions are described in reference 27. The RNA probe was specific for the first exon of cORF.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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HTDV/HERV-K Gag expression depends on posttranscriptional regulation. To investigate whether the expression of HTDV/HERV-K structural proteins is controlled by viral regulatory elements, like HIV expression is regulated by Rev and its responsive RNA element, RRE, we used a well-established HIV-based reporter system with three components: (i) a Rev expression plasmid designated pREV (pBsrev in references 8 and 33) (Fig. 2A); (ii) an RRE sequence-containing HIV Gag expression vector designated pHIVgagRRE (pNLcgagA2 in references 7 and 15) (Fig. 2B); and (iii) HLtat cells (44), a subclone of HeLa cells stably transfected with a Tat expression plasmid. The Gag protein is expressed at high levels only when Rev can bind to the RRE, thereby stabilizing the gag mRNA and enhancing its nuclear export. In transient-transfection assays in HLtat cells, HIV Gag expression can be monitored either by immunofluorescence or by p24 enzyme-linked immunosorbent assay (ELISA).
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)] (Fig. 2A) (for details on plasmid
construction, see Materials and Methods).
When we cotransfected pHIVgagRRE and pREV into HLtat cells, a large
number of HIV Gag-positive cells were detected with a monoclonal
-HIV p24 antibody by immunofluorescence staining (Fig. 2C, panel 1).
Control cotransfections with expression plasmids lacking either the Rev
coding sequence (Fig. 2C, panel 2) or the RRE (data not shown) were
negative for HIV Gag expression. Similarly, when
pHKgagRRE and pREV were transfected into HLtat cells,
HTDV/HERV-K Gag protein was expressed only when Rev was present (Fig.
2C, panel 3); cotransfections of pHKgagRRE and p() (Fig. 2C4) and of
pHKgag and either pREV or p() (data not shown) were negative for Gag
expression as monitored by immunofluorescence using a rabbit or a goat
polyclonal antiserum raised against recombinant HTDV/HERV-K Gag. These
results clearly demonstrate that HERV-K Gag expression depends on
posttranscriptional regulation just as HIV Gag expression depends on a
Rev-RRE-like function. The restriction of the nuclear export of
HTDV/HERV-K Gag may be due to cis-acting elements as for HIV Gag.
cORF and RcRE, the Rev-RRE homologues of HTDV/HERV-K. Having shown that regulatory elements such as Rev and RRE are essential for viral gene expression in the ERV family HTDV/HERV-K, we assumed that cORF was the most probable counterpart to Rev. Using a cORF expression vector (Fig. 3B), we confirmed that, just like Rev, cORF was efficiently expressed independently of posttranscriptional regulation. Therefore, the expression of cORF could also serve as a control for transfection efficiency.
pREV was replaced by pcORF in the test system described above in a first attempt to determine whether cORF could interact with the RRE; however, this experiment failed (data not shown). This was not surprising because many Rev homologues are limited in the use of an unrelated RRE (reviewed in reference 10). Then we searched for the authentic HTDV/HERV-K RRE equivalent by screening the entire 3' one-third of the HTDV/HERV-K genome, starting with nt 6481. The HIV Gag expression vector continued to be used as a reporter, but the RRE sequence in pHIVgagRRE was replaced by consecutive segments of the HTDV/HERV-K genome, overlapping by roughly 200 nt. All of these vectors were cotransfected with either pcORF, pREV, or p() into HLtat cells.
Using immunofluorescence staining as a primary detection method,
significant amounts of HIV Gag were detected only in cotransfections with a reporter plasmid containing the HTDV/HERV-K 3' LTR (U3RU5) (Fig.
3A) as an RRE substitute
and cORF as an effector (data not shown). To define the minimum size of
the cORF-responsive element, smaller fragments of the 3' LTR were
tested (Fig. 3A). We mapped the most efficient fragment to the sequence
U3RIII (nt 8720 to 9148) (Fig. 3A and C and Fig. 3D, panel 1). The
smallest but least efficient cORF-responsive element was mapped to
fragment U3RIX (nt 8720 to 9093) (Fig. 3A and C and Fig. 3D, panel 3).
Cotransfections of a reporter construct carrying the fragment RU5 (nt
9065 to 9492) (Fig. 3A and C and Fig. 3D, panel 5) with pcORF were
negative for HIV Gag expression, as were cotransfections with each of
these reporter constructs and p() (Fig. 3D, panels 2, 4, and 6).
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). In 13 independent transfection experiments, Gag expression was analyzed
either in cell lysates (data not shown) or in particle preparations
purified by ultracentrifugation from the supernatants of
transfected-cell cultures, with equivalent results. A typical experiment, shown in Fig. 3E, clearly indicates that U3RIII and the
minimal fragment U3RIX contain the cORF-responsive element. The
presence of the p55 HIV Gag precursor protein in the positive transfectants was confirmed by Western blot analysis with cell lysates
(Fig. 3F).
The data (Fig. 3E and F) also indicate that Rev may substitute for cORF
on the HTDV/HERV-K sequences, albeit with a much lower efficiency,
confirming observations made in the immunofluorescence studies (data
not shown). Rev function on the smaller element, U3RIX, was no longer
statistically significant, indicating that Rev may bind to a slightly
different sequence. Further analyses will be needed to elucidate this
hypothesis and to map the exact Rev binding site.
In summary, these findings unequivocally demonstrate that cORF has a
Rev-like function. In contrast to HIV, the cORF-responsive element is
not localized in env but lies within the U3R region of the
3' LTR, resembling the Rex-responsive element RxRE in HTLV-1 (1). By analogy, we designate the HTDV/HERV-K element RcRE (for regulatory cORF-responsive element).
Posttranscriptional regulation of HTDV/HERV-K in full-length constructs. To study HTDV/HERV-K gene regulation in the context of the viral genome, we relied on a complete coding sequence (lacking the LTRs) which had recently been constructed from overlapping cDNA clones. This sequence has been shown to induce a high level of Gag expression and the formation of virus-like particles in a baculovirus expression system but does not yield any cORF or Env protein, probably due to inefficient mRNA splicing in insect cells (45).
We used this sequence to construct two expression plasmids which both contain the complete HTDV/HERV-K coding sequence but differ by the presence or absence of most of the 3' LTR and thus in the presence (pHKRcRE) or absence (pHK) of RcRE (Fig. 4A) (for details on plasmid construction, see Materials and Methods). For transfections into primate cell lines in which HTDV/HERV-K mRNAs are not constitutively expressed, like HeLa and COS7 cells, we had to use a foreign promoter, the cytomegalovirus promoter, since HTDV/HERV-K LTRs are active only in teratocarcinoma cell lines (unpublished data). In mammalian cells, in contrast to the baculovirus expression system in insect cells, vectors containing all HTDV/HERV-K genes and splice sites give rise to the formation of cORF transcripts by two splice events (Fig. 1A) (27), alleviating the need to cotransfect pcORF. Again, cORF expression was used to monitor the transfection efficiency.
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Stabilization and enhanced export of incompletely spliced HTDV/HERV-K mRNAs by cORF and RcRE. To confirm the Rev-like function of cORF, we analyzed the nuclear export of HTDV/HERV-K transcripts. pHKRcRE and pHK were transfected into HeLa cells, and nuclear and cytoplasmic mRNAs of the transfectants were prepared. To demonstrate the lack of endogenous HTDV/HERV-K expression, nuclear and cytoplasmic mRNAs from untransfected HeLa cells were prepared in the same way. Equal amounts of nuclear and cytoplasmic mRNAs were analyzed by Northern blotting, using a probe specific for the first exon of cORF, which is present in all predicted mRNA species (a typical experiment is shown in Fig. 5A). HTDV/HERV-K-specific transcripts were consistently detected in the transfected HeLa cells, but not in the untransfected HeLa cells. Viral transcripts comprised the three expected species (Fig. 1A): a full-length transcript, a subgenomic env transcript, and transcripts encoding cORF (the transcripts derived from pHK are 1,050 nt shorter due to the lack of an RcRE). The two cORF bands detected in pHKRcRE transfectants are a consequence of two polyadenylation signals present in the construct, one in the HTDV/HERV-K 3' LTR and the other in the plasmid downstream of the inserted HTDV/HERV-K sequence.
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cORF-mediated nuclear export is CRM1 dependent. Next we studied whether cORF uses the same nuclear export pathway as Rev and Rex, that is, the CRM1-dependent pathway. To investigate this, we took advantage of LMB, a specific inhibitor of CRM1, which very efficiently blocks nuclear export mediated by Rev and Rex (16, 48).
Since LMB is cytotoxic, we determined the optimal concentration of LMB that blocks nuclear export without having toxic effects, using the HIV-based reporter system as an indicator and immunofluorescence as a detection method. We cotransfected pHIVgagRRE or pHIVgagU3RIII with pREV, pcORF, or p() into HLtat cells. When LMB was added to the
transfection and to the growth medium at a concentration of 2 nM,
cORF-RcRE- as well as Rev-RRE-mediated HIV Gag expression was
significantly reduced, while almost no effect on cell proliferation was
seen (data not shown). At a concentration of 4 to 6 nM LMB, cORF-RcRE-
and Rev-RRE-mediated HIV Gag expression was completely blocked (Fig.
6A). Cell viability was examined by
staining for cORF. Using 4 to 6 nM LMB for treatment, the cells
proliferated slightly more slowly, resulting in a smaller number of
cells, but cORF expression in these cells was not dramatically reduced (Fig. 6A, panel 4). In LMB-treated cells, cORF was located in the
nucleoli and no cORF was visible in the cytoplasm, indicating that
nuclear import was not altered but nuclear export was completely inhibited. cORF was preferentially located at the rim of the nucleolus, a phenomenon which has been described for Rev after treatment with LMB
as well (48).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The HTDV/HERV-K family is a unique group of human endogenous retroviruses in so far as they contain open reading frames for all viral proteins necessary for the formation of retroviral particles. Such particles have been detected in teratocarcinoma cell lines (HTDV particles [5, 24-26]). In these cell lines, HTDV/HERV-K are expressed with an RNA pattern reminiscent of that seen for complex retroviruses (26). One of the predominant proteins (cORF), encoded by a small, doubly spliced mRNA, resembles the viral regulatory proteins Rev and Rex in having an arginine-rich NLS and a leucine-rich NES (27). The presented study demonstrates that the cORF protein, interacting with a responsive element in the HTDV/HERV-K sequence, exerts a Rev-like function.
This conclusion is supported by several lines of evidence. (i) HTDV/HERV-K Gag proteins, like HIV Gag proteins, need a Rev-RRE-like export system for proper expression. (ii) For HIV gag-containing constructs, Rev and RRE can be replaced by cORF and a responsive element in the 3' LTR of the HTDV/HERV-K mRNA, as demonstrated by immunofluorescence, determination of p24 levels, and Western blotting. This element, designated RcRE, maps to nt 8720 to 9148 in U3R. (iii) With full-length HTDV/HERV-K constructs, the expression of structural proteins is significantly enhanced by the presence of RcRE. (iv) As shown by Northern blot analysis, the amount of cytoplasmic viral mRNA specific for the structural proteins is also significantly increased when the constructs contain RcRE and when cORF is expressed, demonstrating the stabilization and enhanced nuclear export of these mRNAs by cORF and RcRE. (v) The expression of structural proteins is inhibited by LMB, a potent inhibitor of CRM1-mediated nuclear export. This export pathway has been shown to be used by Rev and Rex (4, 16, 23, 39, 48).
Since RRE and RxRE are complex mRNA structures, the possible existence of similar features in RcRE has been investigated by using the DNASIS RNA secondary structure prediction software. Two adjacent energetically favored stem-loop structures are consistently identified (nt 8839 to 8945). The exact binding sites for Rev and Rex on their responsive elements have been described in detail (see, e.g., references 2, 3, and 22). Rev binds to the sequence 5'UGGGCG/5'CGGUACA, forming a short stem with an internal loop located in stem-loop 2 of the RRE (17). The Rex binding core is mapped to the sequence 5'CUCAGGUCGA(G)/5'(C)UCCCUUGGAG in HTLV-1 (1) and to the sequence 5'GAGCUCG/5'CGCUC in HTLV-2. A putative binding site [5'GGGUCGA(G)/5'CUCCCC] has been identified in simian T-cell leukemia virus (STLV) PH969, a primate T-cell lymphotropic virus isolated from an African baboon (47). In the stem of the first stem-loop structure of RcRE, the sequence 5'CUCCC/5'GGAAGGG shows a striking similarity to the Rex binding domains of the HTLV/STLV group (Fig. 7). Further investigations will aim at high-resolution mapping of the biologically relevant nucleotides to define the core binding sites in RcRE of cORF and of Rev, which seems to be able to replace cORF to some extent. In addition, whether Rex can replace cORF on RcRE or cORF can substitute for Rex on the RxRE will be examined.
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HTDV/HERV-K genomes exist in two subgroups which differ by a deletion of 292 nt at the boundary between the pol and env reading frames. This deletion affects the splice acceptor site for the formation of the subgenomic env transcript and, most importantly, removes the first coding exon of cORF (27). The subclass of proviruses harboring the deletion has been designated type 1 in order to credit the first sequencing of a provirus of this family, the HERV-K 10 provirus (38); the subclass of the prototypic complete genomes has been designated type 2 (Fig. 1A) (27). Type 1 proviruses do not code for the cORF protein but contain RcRE. Therefore, their proper expression may still depend on a cORF-RcRE-based nuclear export system. A survey of whole-cell RNA prepared from different human tissues showed widespread expression of full-length type 1 sequences. However, spliced viral mRNAs or viral proteins could not be detected in those tissues (40). One possible explanation is that the nuclear export of viral RNA is disturbed due to the lack of cORF.
Expression of type 2 proviruses, which encode cORF, is mainly restricted to testicular cells. The precise mechanism leading to the differential expression in testicular cells is not known but may involve a cell type-specific regulation of transcription of putative type 2 specific LTRs (unpublished data). The presence of cORF, translated from type 2 specific mRNA, allows the high-level export of spliced and unspliced viral RNAs into the cytoplasm and their subsequent translation into proteins. Type 1 sequences may contribute to the pool of viral cytoplasmic mRNAs and viral proteins expressed in cases in which cORF is provided in trans. Especially in testicular tumor cell lines, both type 1 and 2 proviruses may be a source for upregulation of viral proteins to a level which allows the formation of particles (24). Surprisingly, HTDV/HERV-K proteins, predominantly cORF, can be detected in normal human testis tissue (40). This finding raises the question of whether cORF has taken on some role in the development of testes in the primate lineage. In this case, the persistence of open reading frames in this class of HERVs would be obvious. It will be interesting to study whether dysregulation of cORF expression contributes to the onset or progression of germ cell tumors.
The particles observed in teratocarcinoma cell lines may be solely Gag particles, because Env is present in only limited amounts in the cells as well as in particle preparations (unpublished data). A similar, albeit less dramatic, imbalance can be seen in transient-transfection assays with a full-length HTDV/HERV-K clone: only 50 to 80% of Gag-producing transfectants express Env. These observations do not conform with the pattern expected for a functional cORF-RcRE interaction and may be explained by as-yet-unidentified deficiencies in HTDV/HERV-K proviruses.
In sequence composition, gag and pol genes of HTDV/HERV-K proviruses resemble those of type D viruses, but their Rev-RRE-like posttranscriptional regulation resembles that of the bovine leukemia virus-HTLV and lentivirus genera. Therefore, these endogenous proviruses could be seen as intermediates in the evolution to contemporary primate retroviruses. Bearing this example in mind, ERVs in general can be regarded as remnants of the evolution buried in the genomes of living cells. Uncovering and analyzing these sequences as fossils may extend our understanding of the different steps taken during the evolution of retroviruses. Our findings, for instance, indicate that the strategy of regulation of gene expression by a viral protein which binds to a responsive element may have evolved 40 million years ago and was not first achieved during the recent evolution of contemporary retroviruses. The observation that exogenous retroviruses frequently recombine with endogenous counterparts may indicate that ERVs provide a useful reservoir of genes by which exogenous retroviruses can evolve new functions. One might even cautiously speculate that an HIV predecessor could have picked up the Rev gene by swapping sequences with an HTDV/HERV-K provirus in the course of evolution. However, other hypotheses, like derivation of HTDV/HERV-K from an even more complex ancestor or the independent evolution of strategies for nuclear export, are still possible.
Having shown the Rev-like function of the cORF protein, the abbreviation of the corresponding gene needs a new interpretation: cORF is convergent to Rev in function.
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ACKNOWLEDGMENTS |
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We thank B. Felber and G. N. Pavlakis, Frederick, Md., for providing plasmids and cells; B. Wolff-Winiski, Novartis, for the gift of leptomycin B; and R. R. Tönjes and J. Denner for providing plasmids and antibodies. We are indebted to U. Held, H. Strobel, A. Hornung, H. Bartel, and H. Rahmouni for excellent technical assistance. We thank K. Boller, M. Chudy, J. Denner, J. Hesse, M. Knößl, R. Kurth, M. Marschall, M. Nübling, R. Seitz, and R. R. Tönjes for helpful discussions. We thank I. Plumbaum for expert help in editing the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany. Phone: 49-6103-773400. Fax: 49-6103-771265. E-mail: loero{at}pei.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, November 1999, p. 9496-9507, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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