Department of Biology and Museum of Zoology,
University of Michigan, Ann Arbor, Michigan 48109-1079
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TEXT |
Endogenous retroviruses have been
found in all vertebrate hosts examined. These viruses are integrated
into host genomes at multiple locations and are usually transmitted
vertically via the germline. The most extensively studied endogenous
avian retroviruses are found in the genome of the domestic chicken and
belong to the avian sarcoma and leukosis virus (ASLV), or
alpharetrovirus, genus. Endogenous ASLVs include Rous associated
virus-0 (RAV-0), endogenous avian retroviruses (EAVs), and avian
retrotransposons from chickens (ART-CH). RAV-0 is thought to represent
a recently integrated endogenous virus, because its provirus DNA
sequence is highly conserved relative to those of exogenous ASLVs such as Rous sarcoma virus (RSV). EAV-0 elements are thought to be older
integrations of avian retroviruses, because they are less similar to
exogenous viruses and their phylogeny closely reflects host phylogeny
(5, 19). The number of known EAVs is increasing and
includes recently identified EAV-HP and ev/J, which are apparently the
same endogenous virus described independently (20, 21). ART-CH elements have deletions in all retroviral genes, but they retain
cis-acting sequences necessary for retrotransposition
(11, 17).
Recently, we showed that endogenous ASLVs are found in three families
of galliform (fowl-like) birds, and, in some cases, the phylogenetic
patterns observed for virus genes were incongruent with host phylogeny
(8). Our findings are beginning to elucidate the ancient
evolutionary association between retroviruses and birds, and they
suggest the possibility of more-recent horizontal transmission of
endogenous viruses between avian hosts as well.
In this report we describe a new avian proviral genome, obtained from a
genomic library of Bonasa umbellus (ruffed grouse), called
tetraonine endogenous retrovirus (TERV). Tetraonines are a subfamily of
galliform birds consisting of grouse and ptarmigan. This is the first
report of a complete, replication-defective ASLV provirus sequence from
a bird other than the domestic chicken. We compare the structure of
TERV to those of published avian retroviruses in order to investigate
its function and evolution. Southern blot and reverse transcription-PCR
(RT-PCR) analyses are used to document the distribution and expression
of TERV-related viruses in galliform birds. We hypothesize that TERV is
an active, endogenous retrovirus formed through recombination between
endogenous retroviral lineages.
Generation of grouse 
bacteriophage genomic library and
characterization of provirus structure.
A B. umbellus
lambda genomic library was constructed using a Lambda FIX
II/XhoI partial-fill-in vector kit (Stratagene, La Jolla,
Calif.). This library was screened by lifting plaques onto nylon
membranes and probing with a 32P-labeled gag
probe. This probe was amplified by PCR using GAG.F1 and GAG.R1 primers
(Fig. 1) and standard PCR conditions as
previously described (8). After gel purification of PCR
products, 
25 ng of probe DNA was radiolabeled using
[
-32P]dATP (3,000 Ci/mmol; Amersham Pharmacia
Biotech), 6 U of Klenow fragments, and random primers. Positive plaques
were grown in liquid culture, and recombinant phage DNA was isolated
using standard protocols (22). Provirus-positive phage DNA
was randomly fragmented and subcloned into pZero vector (Stratagene).
Plasmid DNA was isolated from positive colonies using a QIAprep spin
miniprep kit (Qiagen) and sequenced using universal primers as
previously described (8). The sequence was edited, and
contigs were assembled, using Sequencher (Gene Codes Corp.). Published
ASLV Sequences are listed with GenBank accession numbers in Table
1.
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FIG. 1.
Comparison of RSV (A) and TERV (B) genome and packaging
signal ( ) secondary structures (3, 23). The RSV genome
is not drawn to scale. The first 342 nucleotides (shaded region) of
TERV gag are highly conserved relative to published ASLVs.
The nucleotide similarity ranges from 80 (EAV-HP) to 97% (B. umbellus ASLV). The matrix (MA) region of Gag is indicated above
coding regions. Hatched boxes, three regions in TERV with the greatest
similarity (29 to 59%) to those of EAV-HP (20, 21). The
single line between TERV gag and env denotes an
apparent noncoding sequence. Primers GAG.F1 and GAG.R1
(8), used to amplify the probe for genomic library
screens, are shown below RSV. Predicted secondary structures of the
retroviral packaging sequence were modeled using Mfold (4,
32). Nucleotide positions listed are relative to the 5' end of
the packaging sequence (Fig. 2, M). Major stem-loop structures are
identified using the notation of Banks et al. (3). The RSV
secondary structure is based on a consensus of 20 previously published
ASLV packaging sequences. This structure is identical to the structure
published by Banks and Linial (2).
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The DNA insert of one recombinant lambda was sequenced to reveal a
replication-defective retroviral sequence of 3,711 bp, which we call
TERV (Fig. 2). Host
flanking sequences at the 5' and 3' ends of TERV (1,439 and 733 kb,
respectively) were found to be not similar to any sequences in GenBank
using BLAST (1). The TERV provirus genome structure was
similar to those of other defective avian retroviruses such as Fujinami
sarcoma virus, EAV-HP, and ART-CH (11, 20, 21, 24). The
TERV protein coding sequence was flanked by long terminal repeats
(LTRs), and these in turn were flanked by 5-bp direct repeats
(5'-ATCAG-3'). The terminal nucleotides of the provirus were
5'-TG...CA-3' and were part of imperfect indirect repeats (Fig. 2).
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FIG. 2.
Complete nucleotide sequence of a TERV provirus
including the host flanking sequence. The complete provirus is 3,711 bp
in length. Direct repeats (5 bp) flanking the provirus are shaded, and
major LTR regions are indicated. The repeat region (R),
tRNATrp PBS, and PPT are underlined. The TATA box and
polyadenylation signal are boxed. Translations of Gag and Env proteins
are below the nucleotide sequence. Downward arrow, junction between the
highly conserved gag sequence (5') and regions of low
conservation (3'). The shaded region in Gag corresponds to a region
possibly homologous to the L domain (PPPPY) in ASLVs. Double
underlining, putative packaging sequences (M and direct repeat 1).
Only one copy of RSV-related direct repeat 1 was found in TERV. Primer
sequences described in the text are underlined, and the primer names
are indicated. ProbeMatrix and
Probeenv, fragments representing probes used for
Southern hybridization.
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We compared TERV untranslated region (UTR) sequences to retrovirus UTR
sequences of known function to identify potentially functionally
homologous regions. The 3' boundary of the 5' LTR was distinguished by
a sequence highly conserved relative to those of other avian retrovirus
tRNATrp primer binding sites (PBS) (initiation of
minus-strand synthesis). The 5' boundary of the 3' LTR was
distinguished by the polypurine tract sequence (PPT) (initiation of
plus-strand synthesis). Based on these boundaries, host flanking direct
repeat sequences, and sequence conservation with other ASLV R regions,
identical LTRs of 376 bp were defined. The typical proviral LTR
structure (5'-U3-R-U5-3') was found, and lengths for the U3, R, and U5
regions were 266, 21, and 89 bp, respectively. These LTRs were longer
than LTRs from RSV (327 bp) (23), EAV-HP (315 bp)
(20, 21), EAV-0 (243 bp) (5, 6), and RAV-0
(277) (13) but slightly shorter than ART-CH
LTRs (388 bp) (17).
The TERV U3 sequence was most similar to that of RAV-0, particularly at
conserved motifs that function in retrovirus transcription. Putative
transcription factors were identified by searching on-line database
TRANSFAC, specifying a threshold score of 85 (12), and by
comparative sequence analysis with known transcriptional regulatory
sequences from ASLVs. The 5'-most enhancer binding element was similar
to an IK-2 element (position 
221), which is bound by a transcription
factor isolated from mice (10). The next sequence, a
EFI binding element, was located at 194 in relation to the
transcription start site. It is thought that protein EFI is an
embryonic gene regulator in chickens (9). These two
elements were not known from other avian retrovirus LTRs; thus further
characterization is necessary as these elements show limited similarity
to the consensus sequences. At position 165 there was a putative
serum response element (SRE) (27, 28) that was highly
conserved relative to the SRE found in RAV-0 (31). Within
this element there was a CArG box, defined as
5'-CC(A/T)6GG-3'. This sequence was followed by an inverted
CCAAT box at 132 also found in RAV-0 and RSV. The CCAAT box was in
the middle of putative NF-Y and C/EBP binding sites. Finally, another
SRE was located at 75.
The U3 region of TERV was highly conserved relative to those of other
ASLVs, starting with the TATA box located at position 24, followed by
a polyadenylation signal just upstream of the repeat region (R). The R
and U5 regions of TERV had the greatest sequence similarity to those of
RAV-0 at 91 and 89%, respectively, and had 82 and 86% similarity,
respectively, to those of RSV (Table 2).
The tRNATrp PBS were identical across all avian
retroviruses mentioned in this report with the exception of that of
EAV-0, which had an additional cytosine residue (Table 2).
Analysis of putative Gag and Env proteins.
Two open reading
frames (ORFs) that had sequence similarity to those of published ASLVs
were found. The N-terminal 123 amino acids encoded by the first ORF had
88% identity to RSV and 95 to 97% identity to previously published
B. umbellus Gag sequences corresponding to the M domain of
the matrix region (Fig. 1). The remaining 460 amino acids had 29 to
59% identity with those of ASLV Gag proteins in three regions, each
separated by sequences that showed no significant sequence similarity
to published ASLV amino acid sequences in GenBank (Fig. 1). The regions
of EAV-HP had the highest similarity to these regions, slightly higher
than published regions of B. umbellus ASLV. One other region
of amino acid similarity to Gag begins at P240 and is a
proline-rich region (PSAPSAPPPAP) possibly homologous to the L domain
(PPPPY) found in all ASLVs, which functions in virus assembly
(30).
A second ORF, located 360 bp downstream of the gag gene,
encoded 106 amino acids that had 29 to 40% identity to Gag of ASLV and
murine leukemia virus and porcine endogenous retrovirus Env based on
BLAST search results (Fig. 1). The putative ORF corresponded to the
carboxyl terminus of the transmembrane region of Env. This Env-related
ORF was just upstream of a 110-bp sequence with 89% identity to those
of exogenous ASLVs, which corresponds to the direct repeat 1 sequence
found in all ASLV genomes studied, including replication-defective
transforming avian retroviruses.
Analysis of gag gene transcription.
Sequence
analysis suggests that TERV is capable of transcription. To determine
if gag sequences were transcribed, total RNA was extracted
from heart muscle of one adult B. umbellus animal and whole
8-day-old Phasianus colchicus and Colinus
virginianus embryos using Trizol reagent (Gibco Life Technologies)
according to the manufacturer's protocol. RNA extract was treated with
5 U of DNase I, amplification grade (Gibco Life Technologies), to eliminate DNA contamination according to the manufacturer's protocol. RT-PCR was performed on DNase-treated total RNA using a Titan one-tube
RT-PCR kit (Roche) and two sets of primers at an annealing temperature
of 55°C. Primers GAG.F1 and GAG.R1 (8) are general gag primers, while GC2128F and GC2604R (Fig. 2) are specific
to TERV gag. Chicken 
-actin primers were used as a
positive control for RNA. These primers (-actinF
[5'-AATGAGAGGTTCAGGTGCCC-3'] and -actinR
[5'-ATCACAGGGGTGTGGGTGTT-3']) amplify a 410-bp fragment. RT-PCR products were verified by DNA sequence analysis as described previously (8). PCR using GAG.F1 and GAG.R1 primers was
performed on DNase-treated and untreated samples to verify that DNA
contamination was eliminated.
RT-PCR using GAG.F1 and GAG.R1 primers on DNase-treated samples
resulted in products from all three birds as did PCR analysis of
non-DNase-treated samples (Fig. 3). Size
variation in amplicons is consistent with results obtained in our
previous study of avian retroviral gag sequences
(8). Sequence analysis of the RT-PCR product from
Phasianus suggested that these primers amplified nontarget
transcripts, whereas sequence analysis confirmed that gag
transcripts were amplified from Bonasa and
Colinus. RT-PCR using TERV-specific primers (GC2128F and
GC2604R) resulted in amplification in all three birds. The product from
B. umbellus was 475 bp, the size predicted from TERV, while
the predominant products from Phasianus and
Colinus were 200 to 300 bp larger (Fig. 3). These RT-PCR
products were sequenced, and all were verified as gag
sequences.
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FIG. 3.
RT-PCR analysis of total RNA isolated from heart muscle
for an adult B. umbellus animal (BOUM) and 8-day-old embryos
of P. colchicus (PHCO) and C. virginianus
(COVI). (A) RT-PCR on total RNA. Lane 1, 100-bp ladder (Promega); lanes
2 to 5, primers GAG.F1 and GAG.R1; lanes 6 to 9, primers GC2128F and
GC2604R. (B) RT-PCR using -actin primers (lanes 2 to 5) and PCR
using primers GAG.F1 and GAG.R1 on DNase-treated RNA extracts (lanes 6 to 9). (C) PCR on non-DNase-treated samples using primers GAG.F1 and
GAG.R1.
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Normally, following transcription, two copies of the retroviral RNA
genome are incorporated into viral particles. This packaging process
involves the recognition and binding of sequence on the RNA genome
by viral proteins. In ASLVs, a 160-nucleotide sequence has been
identified as the minimal packaging signal (M) in the leader region
between the PBS and the Gag initiation codon (3). TERV
contained a sequence highly conserved relative to avian retrovirus Ms located between the primer binding site and the gag
initiation codon. TERV M had a similarity of 78.2% to a consensus
alignment (2) of 20 exogenous and endogenous avian
retrovirus packaging sequences. EAV-HP and ART-CH did not have the same
level of conservation in the packaging signal. The sequences between
the PBS and the gag initiation codon in ART-CH and EAV-HP
are about 100 bp shorter than those in TERV and other ASLVs.
It appears that the secondary structure of M plays a significant
role in efficient packaging (2). TERV and the ASLV
consensus packaging sequence were analyzed using the Mfold program
(version 3.0) to model the most-stable secondary structures (4,
16). The lowest free energies were 61.02 kcal/mol for the
secondary structure of TERV and 56.82 kcal/mol for the folding of a
consensus sequence of 20 ASLV packaging signals (Fig. 1). The two
modeled structures were remarkably similar. TERV M had the two major stem-loop regions O3 and L3 and the three minor stem-loops O3SLa, O3SLb, and O3SLc previously identified by Banks and Linial
(2). In ART-CH and EAV-HP the most-stable secondary
structures for sequences that correspond to M were not similar to
those of RSV, the consensus of ASLVs, or TERV when folded with Mfold
(not shown).
Our previous work showing that some ASLV phylogenetic relationships
reflect overlapping host species ranges rather than host species
phylogeny (8) suggests that horizontal transmission of
ASLVs has occurred in the past. Here we have demonstrated that TERV is
transcribed and contains sequences required for packaging and
retrotransposition. We have no direct evidence regarding the means by
which TERV or other tetraonine ASLVs move between host species;
however, one possible mechanism is hybridization among host species,
which is known to occur naturally between some tetraonines (14). Alternatively, TERVs could have been transmitted
horizontally if they were transcribed and packaged with
replication-competent retroviruses.
Phylogeny and recombination.
To explore the relationship of
TERV and other endogenous and exogenous ASLVs, we conducted
phylogenetic analyses using three regions of TERV. Sequence alignments
corresponding to various regions of TERV and ASLVs were performed using
Clustal X (26). Phylogenetic analyses were performed using
maximum parsimony (MP) as implemented in PAUP* (25).
Branch-and-bound MP analyses were conducted, and bootstrap values were
determined using 100 replicate searches.
The first region examined corresponds to the matrix gene of
gag. MP analysis using this region (342 nucleotides) yielded
one tree composed of three major groups (Fig.
4A). The first group consisted of
endogenous and exogenous viruses isolated from birds in the genus
Gallus, including the domestic chicken (Gallus
gallus). The second group consisted of presumably endogenous
proviruses from grouse and ptarmigan (Tetraoninae), and the third group
consisted of endogenous viruses EAV-HP and ART-CH. TERV was the sister
taxon to ASLV, whose gag gene was previously sequenced from
B. umbellus.
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FIG. 4.
Phylogenetic analyses of gag and UTR
sequences. Unrooted MP trees were constructed using the
branch-and-bound option in PAUP* (25). Bootstrap
values along branches were calculated using 100 random-addition
replicate searches. COVI, C. virginianus ASLV; PHCO,
P. colchicus ASLV; LALA, Lagopus
lagopus ASLV; BOUM, B. umbellus ASLV; GAVA, G. varius ASLV; FuSV, Fujinami sarcoma virus; AMV, avian
myeloblastosis virus; Y73, avian sarcoma virus Y73. Branch lengths are
proportional to inferred amounts of evolutionary change. (A)
Relationship of 10 previously published ASLV and TERV sequences based
on 342 nucleotide sites from the matrix region of the gag
gene. (B) One of two equally parsimonious trees for the relationship of
11 published ASLVs and TERV based on the more-divergent amino acid
region of Gag downstream from the region analyzed in panel A. The two
equally parsimonious trees differ only in the placement of GAVA. (C)
Phylogeny of eight previously published UTR sequences and TERV.
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The second region corresponded to the remaining sequence in TERV Gag
adjacent to the highly conserved matrix region. Only short stretches of
amino acids could be aligned to other ASLV Gag proteins, resulting in a
data set with 125 parsimony-informative characters. Two equally
parsimonious trees that differed only in the placement of Gallus
varius ASLV were found (Fig. 4B). Interestingly, TERV and the
other B. umbellus ASLV sequences were not sister taxa based
on this portion of the gag gene. Instead, TERV is located between an EAV-HP/ART-CH group and a group containing the remaining ASLVs.
The third region analyzed was aligned with eight published avian
retrovirus UTR sequences. This phylogeny (Fig. 4C) showed relationships
similar to those from the tree shown in Fig. 4A. We found that EAV-HP
and ART-CH formed a group that was sister to EAV-0 (EAV-0 was not
included in gag phylogeny). TERV was most closely related to
endogenous avian retrovirus RAV-0. No UTR sequences from other
tetraonine retroviruses were available for comparison.
Recombination within the gag gene of RSV can occur with a
relatively high frequency (15). Incongruent trees
from our phylogenetic analyses suggest the possibility that TERV
was formed by recombination between retroviruses. One
recombination point may occur near amino acid 123, where the similarity
of TERV Gag to B. umbellus Gag drops drastically from 97 to
around 40%. Phylogenetic analysis illustrates that this downstream
region of TERV Gag is not sister to those of other tetraonine ASLVs,
RAV-0, or exogenous ASLVs (Fig. 4B). Phylogenetic analysis and amino
acid identity of a short region of Env (transmembrane region) suggest a
relationship that is also incongruent with UTR and 5'
gag phylogenies (not shown). These findings are
compatible with the interpretation that TERV was formed by
recombination, although the parental sequences for divergent regions of
TERV have yet to be discovered. An alternative explanation to
recombination is that highly divergent regions of TERV result from
differing selective pressures on the viral genome. We are examining
additional complete tetraonine retrovirus genomes to investigate this possibility.
Distribution of TERV-related sequences.
To determine the
distribution of TERV-related sequences, genomic DNA was isolated from
six galliform species and one anseriform (Aythya americana)
using standard protocols. Roughly 3 µg of genomic DNA was digested to
completion with HindIII, electrophoresed in a 1.0%
agarose gel, and blotted overnight onto a positively charged nylon
membrane (Hybond-XL; Amersham Pharmacia Biotech). Two different probes,
probematrix and probeenv, were
amplified by PCR from TERV-lambda DNA. These probes corresponded to a
region of matrix (GC2128F and GC2604R) and a divergent region similar
to that in which env is located (GC3877F and GC4332R) (Fig.
2). Hybridization was performed at 65°C in standard buffer overnight
with probematrix first, followed by
probeenv. The nylon membrane was washed in
several steps with decreasing salt and sodium dodecyl sulfate (SDS)
concentrations, down to 0.5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM
NaH2 PO4, and 1 mM EDTA [pH 7.7]) and 0.1%
SDS, and increasing temperature to 65°C. Prior to being probed
with probeenv, the membrane was stripped
by washing with 0.1% SDS at 100°C for 30 min and exposed to film for
1 week to verify that all of probematrix was removed.
Results of Southern analysis at high stringency using both probes were
positive for the five grouse and ptarmigan tested and negative for a
domestic chicken and a redhead (Fig. 5).
Genomic DNA from two B. umbellus individuals was analyzed
and had almost identical banding patterns. Both probes showed complex
hybridization patterns consistent with the idea that TERV integrated in
multiple locations within the bird genome.
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FIG. 5.
Southern blot analysis of
HindIII-digested avian genomic DNA using TERV
probematrix and probeenv.
Hybridization was performed at 65°C, and the filter was washed
with salt-SDS solution at concentrations as low as 0.5×
SSPE-0.5% SDS at 65°C. (A) Probematrix. (B)
Probeenv. Lanes 1 and 2, B. umbellus
(ruffed grouse); lanes 3, Bonasa bonasia (hazel grouse);
lanes 4, Lagopus lagopus (willow ptarmigan); lanes 5, Lagopus leucurus (white-tailed ptarmigan); lanes 6, Dendragapus falcipennis (Siberian grouse); lanes 7, Anseriformes (Aythya americana [redhead]); lanes 8, G. gallus (domestic chicken). This shows the presence of
TERV or closely related elements in all tetraonine birds surveyed.
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Divergence date estimates from fossils suggest that tetraonines
separated from their putative closest extant relative (Meleagris gallopavo [turkey]) in the mid-Miocene (15 to 20 million years ago), while modern tetraonines seem to have been present for at least 1 million years (7, 29). If our limited sampling of birds is
representative, TERV could have integrated into the genome of a
tetraonine sometime during the past 15 million years. Future surveys,
including more Galliformes as well as birds from additional avian
orders, are needed for more-reliable estimates of TERV age and relative
timing of integration into host species genomes. The 100% identity
between TERV LTRs suggests that TERV was active quite recently,
although this activity may have been restricted within the genome.
Nucleotide sequence accession number.
The TERV sequence
obtained in this study has been assigned GenBank accession no.
AF289082.
This work was supported by University of Michigan graduate student
block grant funds to D.E.D. and M.K.
We thank Michael Frohlich, David Parker, and Vici Blanc for excellent
technical assistance and Sergei Drovetski of the University of
Washington Burke Museum for providing some tissue samples used in this study.
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