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J Virol, July 1998, p. 6164-6168, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Human Endogenous Retrovirus Suppresses
Translation of an Associated Fusion Transcript, PLA2L
Paul E.
Kowalski and
Dixie L.
Mager*
Terry Fox Laboratory, B.C. Cancer Research
Centre, and Department of Medical Genetics, University of British
Columbia, Vancouver, British Columbia, Canada V5Z 1L3
Received 18 December 1997/Accepted 9 April 1998
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ABSTRACT |
Human endogenous retroviruses (HERVs) are repetitive, noninfectious
chromosomal elements degenerated from exogenous retroviruses. The
HERV-H family is composed of approximately 1,000 elements which are
dispersed throughout the human genome. We have shown previously that an
HERV-H element splices into a downstream locus, termed PLA2L, which has
a large open reading frame (ORF) containing two domains with
phospholipase A2 homology. Over half of the putative 5' untranslated
region (5' UTR) of the resulting fusion transcript is derived from
HERV-H long-terminal-repeat and internal sequences. As 5' UTRs are
known to modulate translation initiation, we tested for possible
effects upon gene expression at the translation level due to the 5'
fusion with HERV-H sequences. No PLA2L protein was detected in
teratocarcinoma cell lines in which PLA2L mRNA is abundantly expressed.
In addition, despite a high level of transcription, no protein
synthesis was detected when the full-length PLA2L cDNA was expressed in
COS cells. Upon removal of the 5'-terminal HERV-H sequences, PLA2L
protein was seen in transfectants. The 5' UTR contains both small ORFs
and a strong predicted RNA secondary structure, both of which have been
shown to contribute to translation suppression. The HERV-H sequences,
combined with a unique PLA2L 5' UTR sequence, form a predicted RNA
stem-loop that has a stability greater than that proposed to negatively
affect translation. Interestingly, this stem-loop is abolished when the
HERV-H sequences are removed. We hypothesize that the PLA2L 5' HERV-H
sequences function as an abnormally long and complex 5' UTR, resulting
in suppression of translation in both teratocarcinoma cell lines and
full-length cDNA transfectants. This is the first known example of a
endogenous retrovirus integration affecting expression of a
heterologous human gene at the translational level.
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TEXT |
Human endogenous retroviruses
(HERVs) have been estimated to compose approximately 2% of the human
genome by mass (23) and are structurally similar to, and
presumably degenerated from, exogenous retroviruses. While the
retroviral structural genes of most HERVs have largely become
nonfunctional through mutation, the long terminal repeats (LTRs) of
many HERV elements are active and can promote, enhance, splice into,
and polyadenylate adjacent cellular genes (28). The HERV-H
family of elements is most homologous to murine C-type retroviruses
such as murine leukemia virus, and members are found on all
chromosomes, with copy numbers of approximately 1,000 and a similar
number of solitary LTRs per haploid human genome (17).
HERV elements can potentially affect cellular genes by promoting
deletions or translocations due to interelement recombination or by
retrotransposition. Depending upon the location, HERV insertions have
the potential to cause alterations in gene expression such as those
causing alternate tissue specificity, inappropriate promoter activity,
premature truncation of a reading frame via introduction of a
frameshift or alternate polyadenylation (4, 6, 15, 22, 24,
28). By far the most common effects exerted by HERVs upon
cellular genes are at the transcriptional level, not the translational
level. Because the insertion of an HERV into a transcriptional unit is
usually a very disruptive event, the damage is often realized at the
immediate level of transcription, whereas to effect a translational alteration, the damage must be much more subtle and allow transcription to occur.
In order to elucidate the effects that HERV-H elements may exert upon
proximal genes, we have studied a transcript, isolated from a human
teratocarcinoma cell line, that reflects fusion between an HERV-H
element and a novel human locus containing two domains with similarity
to the sequence of secreted phospholipase A2. The HERV-H LTR promotes
this fusion transcript, termed the PLA2L transcript (phospholipase
A2-like transcript), which contains a short segment of the LTR and
leader region of HERV-H sequence spliced to downstream exons. As this
transcript possesses HERV- derived sequences at its 5' terminus, and
as 5' untranslated regions (5' UTRs) of vertebrate genes are known to
regulate the initiation of translation (21), the regulation
of protein synthesis of PLA2L was studied. Here we report that HERV-H
sequences, acting as a 5' UTR, serve to suppress translation of the
PLA2L transcript in both the original teratocarcinoma cell line and in
a heterologous expression system.
PLA2L is a fusion between HERV-H sequences and a novel human locus
whose sequence is similar to that of cellular phospholipase
A2.
The PLA2L transcript was originally isolated from an NTera2D1
human teratocarcinoma cell line cDNA library by a differential hybridization screening procedure. This screen was designed to detect
transcripts that initiated in an HERV-H LTR and subsequently spliced
into downstream cellular genes (5). The PLA2L cDNA isolated
from this screen is 2.4 kb in length, has 251 bp of HERV-H-derived sequence at the 5' end, and encodes a 584-amino-acid product of an open
reading frame (ORF). The ORF is composed primarily of anonymous
sequence, relieved only by two distinct domains with sequences similar
to that of the secreted form of phospholipase A2. Consistent with an
LTR-promoted transcript, the cDNA possesses R and U5 regions of an LTR
at the 5' terminus, followed by the HERV-H leader region, which ends in
the conserved splice donor site (Fig. 1).
In the 5'-terminal 500 bp of the transcript, there are three potential
initiating methionine residues within the same reading frame which
possess Kozak consensus sequences of varied quality. The context of the
first two AUG codons (nucleotides [nt] 101 and 416, respectively) is
suboptimal, especially with regard to the crucial 
3 position, which
is a purine in almost all true initiating codons (12). The
last AUG codon (nt 455) matches the Kozak consensus to a much greater
extent, including both the 3 A and the +4 G, and we consider it to be
the most likely initiation codon.
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FIG. 1.
Schematic of the 5' region of the PLA2L fusion
transcript. HERV-H-encoded sequences, which end at the splice junction
site (SJ; nt 251) are shown as a thick black line. The
HincII site used to delete the 5' HERV-H sequences to create
pPLA2L- del is indicated with a  . Possible initiating methionine
residues are indicated as M1, M2, and M3. The region of the PLA2L cDNA
which was expressed as a GST fusion and used to generate rabbit
polyclonal antibody (PAb) is shown as a black rectangle. The Kozak
consensus sequence aligned with the three potential start codons is
shown within a box. The complete map and sequence of this cDNA have
been published previously (5).
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Endogenous expression of PLA2L in teratocarcinoma cells.
We
showed previously by Northern blot analysis that HERV-H-promoted PLA2L
transcripts are present in NTera2D1, the cell line from which the
original cDNA was isolated, and in an independent teratocarcinoma cell
line, Tera1 (5). The level of PLA2L mRNA is at least 10-fold
higher in Tera1 than in NTera2D1. In Tera1 and NTera2D1, we have no
evidence of PLA2L being transcribed by a promoter other than that of
the HERV-H LTR. Transcription of PLA2L was not detected in other cell
lines by Northern blot analysis or by reverse transcription-PCR.
Interestingly, these results mimic what has been observed for the
population of HERV-H elements in general. HERV-H elements with an LTR
structure like that of the PLA2L element are highly transcribed in
teratocarcinoma cell lines, and of the cell lines tested, Tera1 has the
highest level of HERV-H mRNA (27).
To determine the regulatory role that the HERV-H element may play at
the PLA2L locus, polyclonal antiserum against PLA2L was
raised. A
PLA2L-glutathione
S-transferase (GST) fusion protein
was
generated by amplifying bases 391 to 584 of the original PLA2L
AF-5
cDNA by PCR. This PCR product was cloned into the
SmaI site
of pGEX2T (Pharmacia), in frame to GST. Log-phase bacteria containing
this construct were induced for 5 h at 37°C with 70 µM
fresh isopropyl-
-
D-thiogalactopyranoside
(IPTG; Fisher Biotech) and subsequently lysed by sonication.
Affinity
chromatography with glutathione-agarose beads (Sigma) was
performed
upon the cleared lysate. Agarose beads containing
purified PLA2L- GST
fusion protein were repeatedly washed and were
used, in conjunction
with Freund's incomplete adjuvant, to directly
immunize a New
Zealand White rabbit (
20). Clarified rabbit
anti-PLA2L antiserum
was used in all subsequent Western blotting at a
dilution of approximately
1:750. Prior to the primary boost of
PLA2L- GST protein, preimmune
sera was taken and was determined to
be negative for anti-PLA2L
reactivity. Mammalian cell lysates and
Western blotting were performed
as previously described
(
16).
Translation of the PLA2L mRNA was initially examined by attempting to
detect PLA2L protein in human teratocarcinoma cell lines.
Both NTera2D1
and Tera1 were assayed for the presence of PLA2L
protein synthesis.
However, despite the high level of PLA2L RNA
(
5), no
evidence of specific immunoreactive PLA2L protein was
seen on Western
blots of lysates of Tera1 (Fig.
2) and
NTera2D1
(data not shown) or in immunoprecipitations of Tera1 lysates
with
rabbit polyclonal antiserum (data not shown). Thus, it appears
that PLA2L is either not translated or translated at a much lower
level than the abundance of mRNA suggests.
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FIG. 2.
Anti-PLA2L Western blot of lysates of various cell lines
and transfectants. The Western blot was blocked overnight at 4°C in
2% BLOTTO (5% skim milk powder in phosphate-buffered saline), washed
three times for 1 h each time in TBST (10 mM Tris-HCl [pH 7.4],
150 mM NaCl, 0.05% Tween 20 [Sigma]), and hybridized to rabbit
antiserum in 2% BLOTTO for 1 h. All washing and hybridization
steps, unless noted otherwise in the text, were carried out at room
temperature with constant agitation. The Western blot was then washed
four times for 30 min each time in TBST. The secondary antibody
consisted of horseradish peroxidase-conjugated AffiniPure goat
anti-rabbit immunoglobulin G (Jackson Immunoresearch Laboratories),
which was used at an approximately 1:8,000 dilution in TBST for a
50-min incubation. The Western blot was then washed four times for 30 min each time in TBST and visualized with a Renaissance
enhanced-chemiluminescence kit (Dupont, NEN). Molecular weights (MW)
are noted in thousands.
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HERV-H sequences affect translation of the PLA2L mRNA.
The
lack of detectable PLA2L protein in teratocarcinoma cells which express
the PLA2L mRNA raises the possibility that the presence of HERV-H
sequences in the 5' UTR might inhibit translation. To test
this possibility, translation of different PLA2L cDNA constructs was
examined after transfection into COS cells. The mammalian expression
vector pCDNA3 (Invitrogen) containing the cytomegalovirus early
promoter-enhancer was used as a backbone for two PLA2L constructs. To
construct pPLA2L- Full, the complete 2.4-kb AF-5 cDNA containing
HERV-H sequences at the 5' end (Fig. 1) was cloned into the
EcoRI site of pCDNA3. Since our laboratory has previously
noticed occasional recombination and instability of HERV-H sequences
within the DH5
Escherichia coli strain, this ligation was
transformed into XL2-Blue (Clontech) and STBL2 (Life Technologies), two
independent E. coli strains known to suppress some
recombinations. The constructs derived from XL2-Blue and STBL2
bacteria were termed pPLA2L- Full1 and -Full2, respectively. A
5'-deletion construct, lacking all HERV-H-derived sequences and termed
pPLA2L- del, was generated by inserting the 2,166-bp HincII fragment of the PLA2L cDNA in pBluescript into the
EcoRV site of pCDNA3. The vector-insert junctions of all
constructs were subsequently sequenced to confirm correct orientation
relative to that of the cytomegalovirus promoter. These constructs were transiently transfected into COS cells with DEAE-dextran
(7). Transfected cells were grown for 48 h and then
lysed in Nonidet P-40 lysis buffer as previously described
(16). Following centrifugation, the concentration of the
supernatant was determined by the Bradford assay (Bio-Rad) and
approximately 10 µg of COS transfectant lysates and 20 µg of Tera1
and BaF3 lysates (an irrelevant murine cell line) were
electrophoresed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene
difluoride membrane, and subjected to Western blotting with anti-PLA2L
antiserum. Figure 2 shows immunoreactive products between 65 and 85 kDa
in the pPLA2L- del transfectant and the lack of specific
immunoreactive bands in both the pPLA2L- Full transfectants and the
Tera1 human teratocarcinoma cell line lysates. The faint 43-kDa band
seen in the COS7, BAF3, and Tera1 cell lysates is nonspecific and was seen in all lysates tested, including ones negative for PLA2L transcription as assayed by reverse transcription-PCR. In addition, no
specific immunoreactivity was detected in a negative control murine
hemopoietic cell line, BaF3 (a gift of G. Krystal), or in the
transfectant host cell line, COS. The presence of two to three
immunoreactive bands in the pPLA2L- del transfectant likely signifies either the use of alternative AUGs to initiate translation or
differential glycosylation or modification of the PLA2L protein by COS
cell systems. Proteolytic degradation does not seem to be the cause, as
similar patterns of bands are seen in multiple, independent
transfections (data not shown).
HERV-H sequences suppress PLA2L translation, not
transcription.
To ensure that the observed inhibition of PLA2L
protein synthesis was due to HERV-H sequences inhibiting
translation and not transcription, total RNA was prepared from
all transfectants with Trizol (Life Technologies). RNA
formaldehyde gel electrophoresis and Northern blotting were carried out
as previously described (14) with a 410-bp BbsI
fragment of the PLA2L cDNA containing the first phospholipase A2-like
domain (5) (Fig. 3). Intact, full-length PLA2L message was seen only in the PLA2L transfectant RNAs
and not in the vector control. Figure 3 shows that there is a
slight increase (approximately twofold) in the level of the pPLA2L- del mRNA relative to the level of pPLA2L- Full1 mRNA. This implies either that the deletion of the HERV-H 5' UTR
modestly increases the heterologous transcription of PLA2L or that
transfection of the pPLA2L- del was slightly more efficient.
However, this modest increase cannot account for the absence of
detectable PLA2L protein in the pPLA2L- Full constructs. It is more
likely that the deletion of HERV-H sequences in pPLA2L- del enables
efficient translation of PLA2L protein.
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FIG. 3.
Northern blot of total RNAs from COS cells transfected
with full-length PLA2L cDNA (PLA2L- Full1 and PLA2L- Full2,
respectively), a vector-only control (pCDNA3), and a deletion construct
lacking all HERV-H sequences (PLA2L- del). The lower panel shows the
blot rehybridized to a -actin probe. A 410-bp phospholipase A2
domain probe was labeled with [32P]dCTP (Amersham) and
hybridized to the Northern blot at approximately 3 × 106 dpm/ml for 16 h at 42°C. This Northern blot was
subsequently washed twice for 20 min each time at 55°C in 2× SSPE
(1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])-0.3% SDS, twice for 20 min each time at 55°C in
1× SSPE-0.5% SDS, and twice for 20 min each time at 60°C in 0.3×
SSPE-1% SDS and then exposed to Kodak X-Omat AR film for 48 h at
70°C. A chicken -actin cDNA probe to control for RNA loading was
labeled, hybridized, and washed as described above but was exposed to
X-ray film for only 36 h. Molecular weights (MW) are noted in
thousands.
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The results described here led us to hypothesize that the 5' fusion of
HERV-H sequences to the cellular PLA2L transcript functions
as an
aberrantly long and complex 5' UTR, explaining the concomitant
inhibition of PLA2L protein synthesis. 5' UTRs are known to be
the
primary modulators of translation efficiency because they
control the
initiation of the 43S preinitiation complex, which
contains the
scanning 40S ribosome, on the 5' end of the mRNA.
The 40S ribosome
scans the 5' UTR until an AUG codon in the correct
context
(
13) is found, when the 60S subunit binds the 40S subunit
and translation is initiated. Although the method by which the
40S
ribosome scans the 5' UTR is unknown, certain structures within
the 5'
UTR can greatly repress or inhibit translation (
9).
5' UTRs
which suppress translation generally possess some or all
of the
following features: stable RNA secondary structures such
as
stem- loops, length greater than the average of 100 to 140
bp,
high GC content, and AUG codons with small or micro-ORFs
(µORFs)
upstream of the correct start codon, especially if the
µORF lacks
a termination codon (
12).
If we assume that the AUG codon with the best Kozak consensus is
the true initiation codon, the PLA2L fusion transcript possesses
a
454-bp 5' UTR, with 252 bases being HERV-H derived. This 5'
UTR
contains three µORFs, two of which are in the same reading
frame (+3)
as the correct AUG codon and lack a stop codon. The
third µORF exists
in the +1 frame and contains a stop codon (Fig.
4). µORFs are hypothesized to inhibit
translation by causing stalling
of the scanning ribosome, while lack of
a subsequent stop codon
causes inefficient reinitiation of the 43S
ribosome downstream
at the correct AUG codon (
13).
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FIG. 4.
Various structural elements within the PLA2L 5' UTR. The
5' region of PLA2L cDNA is shown as a black line, HERV-H LTR sequences
are shown as gray boxes, and the junction between HERV-H and cellular
sequences is denoted SJ on the stem-loop. Upstream µORFs are shown
below the line. Potential initiating methionines are underlined and
numbered according to Fig. 1. The putative start of translation is
shown by a bent arrow. The strongest and most stable predicted RNA
stem-loop is shown above the line, from nt 209 to 369, with a
G of 52.1 kcal/mol. The HincII site used to
construct pPLA2L- del is shown on the RNA stem-loop.
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Additional potential encumbrances to translation which the PLA2L 5' UTR
possesses are predicted secondary RNA structures such
as stem-loops.
These structural elements may be the most potent
translational
inhibitory element found in 5' UTRs (
8,
29).
5' UTRs
containing secondary structures with a free energy of
greater than
30
kcal/mol are known to obstruct the scanning of
an mRNA by the 43S
preinitiation ribosome (
11). While prediction
methods
differ, the energy minimization method of Zuker (
30)
used by
the RNAStructure program (
19) predicts a strong stem-loop
with a free energy of
52.1 kcal/mol, in the PLA2L 5' UTR, between
nt
209 and 369. The probability that this predicted stem-loop
functions in
the observed translational suppression of PLA2L is
supported by the
observation that the
HincII site used to delete
HERV-H
sequences and construct pPLA2L- del occurs in the center
of this
stem-loop. Removal of sequences 5' to the site would destroy
the
predicted stem-loop (Fig.
4). The GC nucleotide content of
the PLA2L 5'
UTR does not appear to differ from the average.
Although deletion of the HERV-H-encoded 5'-terminal 251 bases releases
the PLA2L transcript from translational suppression,
allowing efficient
heterologous protein synthesis (Fig.
2), unique
5' UTR sequences
proximal to the junction with HERV-H also play
a crucial role in
translational control. Insertion of the 251-bp
HERV-H fragment
into the 5' UTR of an unrelated reporter gene
(the human
cell surface molecule Thy-1/CD90 [
3]) within the
same
vector resulted in no change in protein expression, relative
to
that of controls (data not shown). This indicates that the
HERV-H
fragment does not adversely affect the translation of all
genes.
These results suggest that both the juxtaposition of proximal
sequences
unique to the PLA2L locus and HERV-H sequences are necessary
for the
inhibition of PLA2L protein synthesis. This phenomenon
is predicted by
the RNA stem-loop seen in Fig.
4, which is composed
of both HERV-H and
unique PLA2L sequences.
Transcriptional effects of endogenous retroviruses on cellular genes
are common, with numerous examples being reported for
mice and some
for humans (
1,
26,
28). At the PLA2L locus,
we demonstrated
previously that the HERV-H element appears to
have assumed
transcriptional control of the region in teratocarcinoma
cells where
HERV-H LTRs are highly active (
5). In addition,
we have
recent evidence suggesting that the PLA2L transcripts
produced in these
cells are actually fusions of HERV-H with two
unrelated downstream
genes (unpublished data). Translational effects
of retroviruses on
cellular genes are much less common, but a
few cases have been
reported. In a murine lymphoma line, it has
been found that an
exogenous Moloney murine leukemia retrovirus
inserts into the 5' UTR of
the
lck proto-oncogene, leading to
downregulation of
translation (
18). Similar to our PLA2L results,
the
suppression is removed upon deletion of the retroviral sequences
from the 5' UTR. In contrast to what is known about suppression,
two
examples of translational activation due to 5' UTR insertion
of
exogenous retroviruses are known. In the first, the only other
known
example for a human cell line, interleukin 15 protein synthesis
is
increased in a T-cell leukemia line due to human T-cell leukemia
virus
type 1 integration in the 5' UTR of the interleukin 15 gene
(
2). A similar event in a murine leukemia line, due to a 5'
UTR murine leukemia virus insertion, results in upregulated
translation
of the c-
akt proto-oncogene (
25).
In this study we have shown that HERV-H sequences suppress translation
at the PLA2L locus. To our knowledge, this appears
to be the first
description of a retroviral insertion (endogenous
or exogenous)
affecting the translation of a human transcript.
Interestingly,
the transcriptional and translational effects mediated
by the HERV-H
element are presumably not detrimental to the species,
since this
particular HERV-H insertion has been fixed in the primate
germ
line for 15 to 20 million years (
10). The PLA2L fusion
transcript studied here has been detected only in teratocarcinoma
cells
where the LTR promoter is most active (
5). The HERV-H
element appears to be within an intron, so it is possible that
a native
promoter located 5' to the retroviral element is active
in other cell
types, resulting in removal of the entire HERV-H
element by splicing.
Unfortunately, we do not know the function
of the gene into which the
HERV-H element has inserted, since
it has no strong similarity to known
genes and no part of the
PLA2L transcript is yet represented in the
human Expressed Sequence
Tag database (as of March 1998). However,
while the functional
significance of the finding reported here remains
unknown at this
time, it illustrates a novel way in which retroviral
insertions
can affect gene expression.
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ACKNOWLEDGMENTS |
We thank Robert Kay, Gerald Krystal, and Mark Ware (Terry Fox
Laboratory) for kind assistance with protein and fluorescence-activated cell sorting techniques and Patrik Medstrand for critically reading the
manuscript.
This work was supported by a grant from the Medical Research Council of
Canada.
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FOOTNOTES |
*
Corresponding author. Mailing address: Terry Fox
Laboratory, B.C. Cancer Research Centre, 601 W. 10th Ave., Vancouver,
British Columbia, Canada V5Z 1L3. Phone: (604) 877-6070, ext. 3185. Fax: (604) 877-0712. E-mail: dixie{at}unixg.ubc.ca.
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J Virol, July 1998, p. 6164-6168, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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