Previous Article | Next Article 
Journal of Virology, May 1999, p. 4327-4340, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Structures of Endogenous Nonecotropic Murine
Leukemia Virus (MLV) Long Terminal Repeats in Wild Mice:
Implication for Evolution of MLVs
Keizo
Tomonaga and
John M.
Coffin*
Department of Molecular Biology and
Microbiology, Tufts University, School of Medicine, Boston,
Massachusetts 02111
Received 29 October 1998/Accepted 13 February 1999
 |
ABSTRACT |
To develop a better understanding of the interaction between
retroviruses and their hosts, we have investigated the polymorphism in
endogenous murine leukemia proviruses (MLVs). We used genomic libraries
of wild mouse DNAs and PCR to analyze genetic variation in the
proviruses found in wild mouse species, including Mus
musculus (M. m. castaneus, M. m.
musculus, M. m. molossinus, and M. m. domesticus), Mus spretus, and Mus
spicelegus, as well as some inbred laboratory strains. In this
analysis, we detected several unique forms of sequence organization in
the U3 regions of the long terminal repeats of these proviruses. The
distribution of the proviruses with unique U3 structures demonstrated
that xenotropic MLV-related proviruses were present only in M. musculus subspecies, while polytropic MLV-related proviruses were
found in both M. musculus and M. spretus.
Furthermore, one unique provirus from M. spicelegus was
found to be equidistant from ecotropic provirus and nonecotropic
provirus by phylogenetic analysis. This provirus, termed HEMV, was thus
likely to be related to the common ancestor of these MLVs. Moreover, an
ancestral type of polytropic MLV-related provirus was detected in
M. spretus species. Despite their "ancestral" phylogenetic position, proviruses of these types are not widespread in
mice, implying more-recent spread by infection rather than inheritance.
These results imply that recent evolution of these proviruses involved
alternating periods of replication as virus and residence in the germ line.
| |
INTRODUCTION |
All inbred laboratory strains of
mice contain numerous endogenous proviruses, of which those related to
murine leukemia virus (type C MLVs) are the best-characterized group.
Endogenous MLVs are divided into two major groups, ecotropic and
nonecotropic, classified by their potential host ranges (4).
Ecotropic proviruses are present in one to five copies in some, but not
all, common laboratory mouse strains (26, 30, 40).
Nonecotropic viruses are subdivided into three major groups, termed
xenotropic, polytropic, and modified polytropic, and are present in
about 20 copies each in the genome of inbred mice (21, 30,
50). Many of these proviruses have been chromosomally mapped in
laboratory strains, and several have been molecularly cloned and
sequenced (3, 7, 19, 20, 21, 23, 27, 28, 32, 37). These
studies demonstrated that the nonecotropic MLV proviruses are highly
polymorphic in their insertion sites and exhibit limited genetic
variation from one provirus to the next. Our previous studies have
shown that the members of each group of nonecotropic proviruses share a
set of linked polymorphisms in env and the long terminal
repeat (LTR) regions that distinguishes them from the members of other groups (12, 49). Most usefully, the polymorphisms allowed us
to develop a set of oligonucleotide probes that unambiguously detect
members of each nonecotropic group in the mouse genome (18,
50).
The nonecotropic proviruses are more widely distributed than the
ecotropic proviruses and are also found in wild mouse species, especially Mus musculus, which are the progenitors of common
inbred laboratory strains (7, 25, 26, 30, 54, 55, 63). The
distribution of the nonecotropic proviruses in taxonomically distinct
wild mouse species indicates that these germ line sequences were
acquired independently and have remained largely segregated in
Mus (30, 55). Either interbreeding between
different subspecies or cross-species infection could have contributed
to this spread. Some of the nonecotropic proviruses in wild mice
have been cloned and analyzed (7, 55). Recently, we have
demonstrated that, although in common laboratory strains the linkage of
group-specific sequences of the proviruses is strict, proviruses that
combine env and LTR sequences from different groups are
commonly observed in M. musculus subspecies
(55). Furthermore, we have found extensive genetic variation
of nonecotropic proviruses in the wild mice (55). These
characteristics of the endogenous nonecotropic MLV proviruses provide
better understanding not only of the host-retrovirus interaction but
also of coevolution of MLVs and their murine host. Furthermore, because
MLVs have survived as both viruses and endogenous proviruses
in their murine hosts, analysis of the polymorphism of the
nonecotropic MLV proviruses could give us a good way to understand adaptation of the MLVs to the hosts. In fact, the presence of endogenous proviruses that have undergone recombination with other
endogenous viruses in wild mice implies that the endogenous proviruses
have also adapted as viruses in their hosts (55).
We describe here a systematic investigation of polymorphism of
nonecotropic MLV proviruses in wild mice, including M. musculus subspecies (M. m. castaneus, M. m.
musculus, M. m. molossinus, and M. m.
domesticus), Mus spretus, and Mus spicelegus
(formerly known as Mus hortulanus), as well as some inbred
laboratory strains. Using genomic libraries of wild mouse DNA and
specific oligonucleotide probes, we detected several unique U3 regions
of nonecotropic proviruses and determined their detailed distribution
in the wild mice. Although most of the structures were found only in
M. musculus subspecies, some were distributed in both
M. musculus and other distinct species. Furthermore, we
could detect possible ancestral forms of the nonecotropic MLVs. This
paper reports a possible evolutionary relationship between MLVs and
wild mouse species.
| |
MATERIALS AND METHODS |
DNAs.
In addition to four common inbred laboratory strains
(AKR/J, HRS/J, C3H/HeJ, and C57BL/6J), DNAs from several species of
Mus were used in this study. The DNAs of CAST/Ei,
CASA/Rk (M. m. castaneus), CZECH II/Ei (M. m. musculus), MOLC/Rk, MOLF/Ei, MOLG/Dn (M. m. molossinus), WSB/Ei, ZALENDE/Ei (M. m. domesticus),
SF/CamEi, PERA/Rk, PERC/Ei (M. musculus), SPRET/Ei
(M. spretus), and PANCEVO/Ei (M. spicelegus)
strains were obtained from the Mouse DNA Resource of The Jackson
Laboratory, Bar Harbor, Maine. Samples of M. spicelegus (Halbturn), Mus cervicolor, Mus
caroli, and Mus cookii DNA were kindly supplied by
Christine A. Kozak of the National Institute of Allergy and Infectious
Diseases, Bethesda, Maryland.
Preparation of genomic libraries.
DNAs from the CZECH II/Ei
(M. m. musculus) and MOLC/Rk (M. m. molossinus)
strains were partially digested with the restriction enzyme
BamHI. The DNAs were then ligated into the BamHI
site of lambda EMBL3 vector (Stratagene), packaged, and subsequently
amplified in XL1-blue MRA-P2 cells. The library was plated out and then screened by using a 5'-end 32P-labeled xenotropic
MLV-related or polytropic MLV-related (KT-69) oligonucleotide probe.
The xenotropic virus-related probe (Xltr) is described elsewhere
(18). Positive clones were selected and purified. Some of
the unique clones were subcloned into the BamHI site of the
pBluescript SK(
) plasmid vector (Stratagene) and sequenced.
Dried gel hybridization and oligonucleotide probes.
Hybridization in dried agarose gels (unblotting) was described
previously (51). Briefly, genomic DNA digested with
appropriate restriction enzymes was electrophoresed in a 0.8% agarose
gel. After staining with ethidium bromide (EtBr), the DNA in the gel was denatured. After drying, the gel was hybridized for 16 h with a 5'-end 32P-labeled oligonucleotide probe (0.5 × 106 cpm/ml). The dried gel was then washed, briefly air
dried, and exposed to X-ray film for 1 to 5 days by using an
intensifying screen at 70°C. Sequences of oligonucleotide probes
specific for each nonecotropic provirus LTR region (Xltr, Pltr, and
Mltr) and the hybridization temperatures are described elsewhere
(18). Sequences of oligonucleotide probes specific for other
types of the nonecotropic proviruses (and the hybridization
temperatures) are as follows: KT-51, 5'-TAC TAG GAC AAG GGC CAA ACA
GG-3' (62°C); KT-53, 5'-GAC AAG GGC CAA GAA CCG ATG GTA C-3'
(62°C); KT-55, 5'-GGA TAT CTG TGG TCG AGC ACC TGG-3' (60°C); KT-58,
5'-GGC TGA ATA GGT ATC GGT GGT-3' (58°C); KT-59/60, 5'-GGA AGT TCA
GTT A(G/A)A GAT CAA GGC TG-3' (60°C); KT-61, 5'-GCC ATA AGC AAG CTA
GCA ATA GTA AC-3' (60°C); KT-69, 5'-GAA CCA GCA ACA GAC ACA GAA G-3'
(59°C); and KT-76, 5'-GCT GCC ATT TTG CAA GGC ATA G-3' (59°C).
Synthetic oligonucleotide primers and PCR analysis.
To
detect nonecotropic MLV proviruses in mouse genomes by PCR, several
amplification primers were generated in this study. The nucleotide
sequences of the primers are as follows: Uniltr-4, 5'-CGG GCG ACT CAG
TCT ATC GG-3'; KS-50, 5'-CAG TAT CAC CAA CTC AAA TC-3'; Unienv-3,
5'-GGA TAC ACG CCG CTC ACG TA-3'; HE-8, 5'-CTA CAG AAC CGT AGA GGA
CT-3'; and KA-59C, 5'-CAG CCT TGA TCT CTA ACT GAA CTT CC-3'. The
locations of these sequences on the proviruses are shown in the
relevant figures. PCRs were carried out in a total volume of 50 µl
containing 0.5 µg of genomic DNA, 50 pmol each of sense and antisense
primers, and 2.5 U of Thermus aquaticus DNA polymerase
(Taq polymerase; Perkin-Elmer Cetus). The reaction mixtures
for amplification were incubated at appropriate temperatures, and the
cycle was repeated 30 times in a GeneAmp PCR system 2400 (Perkin-Elmer Cetus).
Cloning and sequencing analysis.
The endogenous provirus
fragments detected by PCR were purified from agarose gels and blunt
ended by T4 DNA polymerase (New England Biolabs, Inc.). The fragments
were then cloned into the SmaI site of the pUC119 vector.
DNA sequences were determined in accordance with the double-stranded
dideoxy-chain termination method (45) by using the Sequenase
version 2.0 kit (United States Biochemical Co.).
Sequence alignments and phylogenetic analysis.
Sequences
were aligned by using the algorithm of Needleman and Wunsch
(39) as implemented in the PILEUP program in the Genetics Computer Group (GCG) program (14). For phylogenetic
analysis, genetic distances between pairs of the sequences were
calculated by the DNADIST or PROTDIST program in PHYLIP version 3.5 (16), using Kimura's two-parameter model (29). A
neighbor-joining tree was estimated by NEIGHBOR program and a bootstrap
analysis (15) using 1,000 bootstrap replications to assess
the support at each of the internal nodes of the neighbor-joining tree.
Nucleotide sequence accession numbers.
Provirus sequences
reported in this study have been deposited in GenBank under accession
no. AF070719 to AF070732.
| |
RESULTS |
Our previous study demonstrated that nonecotropic MLV proviruses
show extensive genetic variation in wild mice (55). In particular, U3 regions of the proviruses are highly polymorphic, reflecting evolution of the MLVs in the mice. To investigate genetic variation of U3 regions in the endogenous nonecotropic MLV
proviruses in more detail, we generated genomic libraries from
M. m. musculus and M. m. molossinus DNAs. To
analyze unique U3 forms of the nonecotropic proviruses, the
libraries were screened by using xenotropic or other
type-specific oligonucleotide probes, and individual fragments were
selected and subcloned (see Materials and Methods). A number of
clones containing either the 5' or the 3' LTR sequences of nonecotropic
MLV proviruses were selected for analysis.
In addition to genomic libraries, U3 regions of the proviruses were
also cloned following PCR amplification. For the PCR, we designed an
antisense primer, Uniltr-4, in the R region of the LTR that
annealed to all groups of nonecotropic proviruses and a sense primer,
KS-50, derived from a conserved region near the 5' end of the LTR. We
used this primer pair for amplification of U3 regions from wild mouse
DNAs. The exact sequences and locations of the primers are shown
in Materials and Methods and Fig. 1, respectively. Amplified products were cloned into a plasmid
vector and then sequenced. We have analyzed a total of 55 U3 clones
from M. musculus and M. spretus species,
designated by the prefix Mxv, Mcv, or SPR (Table
1). U3 sequences of unique clones were
aligned with those of previous isolates of MLVs. Although U3 sequences of the nonecotropic proviruses are highly conserved, reflecting their
recent evolutionary relationship, the proviruses could be clearly
classified by unique structural features resulting from duplication,
deletion, or point mutation of the sequences.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 1.
Structure of the MLV LTR. The unique sequences in the U3
region are indicated. The locations of primers and probes used in this
study are also shown. The numbering of the boxed regions corresponds to
that in our previous study (55). UCR, upstream conserved
region (17); DR, direct repeat.
|
|
The sequence regions in U3 useful for classification of the structures
are indicated in Fig. 1. The numbers of the boxed regions correspond to
those in our previous study (55). The 5' end is the most
conserved site among known MLV U3 regions. It includes a
PstI recognition site and the upstream conserved region
(UCR) motif, CGCCAT (17). The approximately
100-bp sequence following region 1 contains a number of
core-enhancer binding sequences that are well conserved among
nonecotropic proviruses. Some MLVs contain a direct repeat of the
enhancer-rich region region 4. The 93-bp sequence immediately 3'
to region 4 includes several transcriptional factor binding sites
and is called the downstream-of-the-enhancer (DEN) region
(57). This region has been shown to play an important role
in transcriptional activation of the LTR (10, 57).
Characteristically, the polytropic MLV-related proviruses contain a
unique 190-bp insertion in the 5' end of the DEN region. The U3 regions
of MLVs also contain both CAT and TATA promoter-associated motifs in
the 3' ends of the sequences.
In the following paragraphs, we describe some specific features
of these regions, which are useful for classifying the various provirus types and understanding their evolutionary relationships.
Structure of endogenous xenotropic MLV-related provirus U3
regions.
The xenotropic MLV-related proviruses could be divided
into four subgroups, termed X-I to X-IV (Fig.
2). The type X-I proviruses include the
previously identified proviruses Bxv1, NFS, and CWM, which
encode infectious xenotropic virus (28, 35). Bxv1
is also involved in the generation of oncogenic mink cell focus-forming (MCF) viruses in certain mice (23). The X-I proviruses
differed from the others by the presence of only one copy of region 1 as well as the enhancer-rich region 4. This group was also
distinguished from the others by a number of single-base differences,
some of which affected enhancer motifs.
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 2.
U3 regions of xenotropic MLV-related proviruses. U3
sequences were cloned from M. musculus DNAs following
amplification with the KS-50 and Uniltr-4 primers. Sequences which were
potentially reactive the Xltr probe and which lacked the 190-bp insert
are aligned with those of several previously sequenced provirus
isolates: NFS (NFS-Th-1) (28), CWM (CWM-S-5X)
(35), Bxv1 (23), Xmv44
(52), MX30 (49), Xmv28 (6), MX27, and
MX33 (49). The sequence of NFS xenotropic provirus was used
as a standard. Dots indicate nucleotide identity. Dashes indicate
absence of a nucleotide. Direct repeats and unique sequences present in
the proviruses are boxed. Potential enhancer sequence regions and
transcriptional factor binding sites are indicated by the shaded and
black bars, respectively. Locations of two promoter-associated motifs,
the CAT and TATA boxes, are also indicated. The position of the 190-bp
insertion in polytropic virus-related proviruses, MX27 and MX33, is
shown by an arrow. The sequences of oligonucleotide probes used for
unblotting analysis are underlined. The conserved PstI
recognition site is also shown. The size of the U3 region of each
provirus is indicated at the end of the sequence in parentheses.
|
|
Type X-II and X-III xenotropic MLV-related proviruses were
isolated from
M. m. musculus and
M. m.
molossinus, respectively,
as well as inbred laboratory
strains. The structures of X-II and
X-III were virtually identical to
each other (Fig.
2). Both proviruses
contained a 14-bp duplication of
region 1 in addition to region
1 (1* and 1), and both types included
common mutations in several
regions, as well as a common insertion
located 12 bp upstream
of the CAT box. The X-III proviruses were
distinguished by a 45-bp
deletion in the middle of the enhancer region,
resulting in the
absence of CArG, LVb, and core-binding enhancer
motifs. The deletion
probably occurred by recombination
between the 7-bp direct repeats,
GGGCCAA, found at
both ends of the deleted sequence in the X-II
proviruses. Furthermore,
half of the type X-III clones we examined
contained an additional
direct repeat of region 4 (data not shown).
In addition, one X-II
sequence,
Xmv44 (
52), contained an 11-bp
deletion
in the small region (region 5)
repeat.
A few clones from the
M. m. musculus library were classified
as type X-IV. The U3 regions of these proviruses had the same
structure
as type A Poly/Xeno recombinant proviruses isolated
from
M. m.
musculus and
M. m. domesticus (
55). Although
the
U3 structure of this type of provirus was similar to that of the
X-I proviruses, several common mutations found in the X-I proviruses
were not conserved in type X-IV proviruses. For example, the region
1 sequence was different from that of the X-I provirus (Fig.
2).
Furthermore, the sequences of both enhancer and DEN regions clearly
distinguished the X-IV proviruses from the X-I proviruses.
Interestingly,
this type of provirus was very similar to polytropic
proviruses
in the 3' end of the U3 region, including the CAT
box.
Structure of endogenous polytropic MLV-related provirus U3 regions
in wild mice.
We next analyzed the U3 regions of polytropic
MLV-related proviruses (Fig. 3). Since
previous studies showed that M. spretus species also carry
polytropic MLV-related proviruses (7, 30), we also isolated
proviruses from M. spretus as well as M. musculus DNAs. U3 clones from M. spretus species were named with
designations beginning with SPR. All proviruses contained a unique
190-bp insertion characteristic of this group (49). Despite
the similarity of these proviruses, they could readily be subdivided
into several types.
View larger version (53K):
[in this window]
[in a new window]
|
FIG. 3.
Alignment of U3 sequences of polytropic MLV-related
proviruses. Sequences of polytropic MLV-related U3 regions cloned from
M. musculus and M. spretus DNAs as described in
the previous legend are aligned with those of polytropic (MX27) and
modified polytropic (MX33) proviruses (49). The MX27
sequence was used as a standard. Dots indicate nucleotide identity.
Dashes indicate absence of a nucleotide. Direct repeats and unique
sequences present in the proviruses are boxed (55). The
190-bp inserted region is also boxed. Potential enhancer sequence
regions are indicated by the shaded bar. Locations of two
promoter-associated motifs, the CAT and TATA boxes, are also indicated.
The sequences of oligonucleotide probes are underlined. The conserved
PstI recognition site is also shown. The size of the U3
region of each provirus is indicated at the end of the sequence in
parentheses.
|
|
The structures of type P-I and P-II proviruses were identical to those
of proviruses isolated from inbred laboratory strains
and previously
classified as polytropic (MX27) and modified polytropic
(MX33),
respectively (
49). Both types were also found in
M. spretus. The P-I and P-II proviruses shared a 14-bp duplication
of
region 1. P-I proviruses also differed by two deletions from
other
sequences. The lack of region 2 resulted in deletion of
the CArG and
LVb enhancer sequences. Furthermore, there was a
49-bp deletion in the
variable 5' boundary region between the
190-bp insertion and original
viral sequences where the P-II proviruses
had an 11-bp deletion
relative to the apparently complete P-III,
-IV, and -V sequences. Both
types lacked the region 5* duplication
but contained a complete
duplication of region 6 (Fig.
3).
Mxv4 was isolated from the
M. musculus library and
classified as a type P-III provirus, although its U3 region was
virtually
identical to that of the P-II proviruses, since it lacked
some
nucleotide changes commonly found in the P-II proviruses and it
carried an "intact" 190-bp inserted sequence. Type P-IV and P-V
proviruses were detected in both
M. musculus and
M. spretus DNAs.
The 5' half of the P-IV U3 sequence had the same
structure as
type B Xeno/mPoly recombinant proviruses from
M. m.
domesticus (
55). The sequences of the P-IV and P-V U3
regions were almost
identical to each other but contained
distinguishing deletions
in the 5' portion. As with the P-I proviruses,
the 23-bp deletion
in the P-IV proviruses resulted in the absence of
CArG and LVb
enhancer motifs. Both types also contained intact 190-bp
insertions
in the sequences. Interestingly, unlike other polytropic
virus-related
proviruses, both proviruses lacked the 1* and 6*
duplications
but contained the region 5* duplication. Moreover, the CAT
boxes
of these types resembled those of type X-II and X-III proviruses
rather than the other polytropic MLV-related
proviruses.
Distribution of nonecotropic MLV proviruses.
To investigate in
detail the distribution of nonecotropic MLV proviruses in wild mice, we
used structural features unique to each U3 type to design specific
oligonucleotide probes. The locations of the probes are shown in Fig. 2
and 3, and their exact sequences are listed in Materials and Methods.
Using these probes, we analyzed PvuII-digested genomic DNAs
from the wild and laboratory mouse strains listed in Table
2. The approximate number of fragments reactive with each probe is also indicated in Table 2. Because of the
similarity of the U3 sequences of some proviruses, some probes detected
more than one type. For example, a probe used for detection of X-IV
proviruses, KT-59/60, could also hybridize to the P-IV and P-V
proviruses (Fig. 2 and 3). In this case, we determined the number of
the X-IV provirus by subtracting the numbers of P-IV and P-V
proviruses from those of KT-59/60 reactive fragments. As
shown in Table 2, wild mice contained large and variable numbers of
proviruses.
The X-I, X-II, and X-III proviruses showed similar distributions in the
wild mouse species. These proviruses were found only
in
M. musculus subspecies (Fig.
4A to C
and Table
2). Although
the probe specific for the X-I provirus detected
a few fragments
in
M. m. domesticus DNAs (Fig.
4A, lanes j,
and Table
2), the
dominant carriers of the X-I and X-II
proviruses were
M. m. musculus and
M. m.
molossinus (Fig.
4A and B, lanes f to i, and Table
2)
with more
than 50 fragments each in the latter DNA.
M. m. molossinus DNAs also had a large number of X-III fragments, but only 12 fragments
weakly reactive with the probe were found in
M. m. musculus
DNA
(Fig.
4C, lane f, and Table
2). By contrast, X-IV-reactive
proviruses
were found in only two distinct species,
M. musculus and
M. spicelegus (Fig.
4D and
Table
2). The X-IV probe (KT-59/60) also detected
fragments in
M. spretus DNA (Fig.
4D, lanes o), but these bands
belonged to either
type P-IV or P-V proviruses (see below, Fig.
5C and D, lanes a to c).
Comigration of the fragments found in
the two different
M. spicelegus strains (PANCEVO/Ei strain and
Halbturn) (Fig.
4D,
lanes p and q) implies that this endogenous
provirus is widespread in
this species. Furthermore, the two X-IV
fragments found in
M. m.
molossinus species comigrated with those
of two laboratory strains
(Fig.
4D, lanes b, d, and g to i).
View larger version (75K):
[in this window]
[in a new window]
|
FIG. 4.
Distribution of xenotropic MLV-related proviruses in
wild mice. Analysis of PvuII-digested mouse DNAs was
performed by hybridization of dried agarose gels with an X-I-specific
oligonucleotide probe (KT-55) (A), an X-II-specific oligonucleotide
probe (KT-51) (B), an X-III-specific oligonucleotide probe (KT-53) (C),
and the X-IV-reactive oligonucleotide probe (KT-59/60) (D). Lanes (a to
d contain laboratory [lab.] strains): a, AKR/J; b, HRS/J; c, C3H/HeJ;
d, C57BL/6J; e, CAST/Ei (M. m. castaneus) (m.m.cas.); f,
CZECH II/Ei (M. m. musculus) (m.m.mus.); g, MOLC/Rk
(M. m. molossinus); h, MOLF/Ei (M. m.
molossinus); i, MOLG/Dn (M. m. molossinus) (m.m.mol.);
j, WSB/Ei (M. m. domesticus); k, ZALENDE/Ei (M. m.
domesticus) (m.m.dom.); l, SF/CamEi (M. musculus); m,
PERA/Rk (M. musculus); n, PERC/Ei (M. musculus) (m.mus.); o, SPRET/Ei (M. spretus) (m.spr.);
p, PANCEVO/Ei (M. spicelegus); q, Halbturn (M. spicelegus) (m.spi.); r, M. cervicolor (m.cer.); s,
M. caroli (m.car.); t, M. cookii (m.coo.). Some
known provirus loci, Bxv1 (23), Xmv10,
and Xmv28 (19), are shown by arrows. The
approximate positions of molecular markers are also shown.
|
|
Almost all the fragments reactive with the xenotropic MLV-related
provirus probes could be detected by the more generalized
xenotropic
MLV-related provirus LTR probe, Xltr (Fig.
4E) (
55),
because
all types of the xenotropic MLV-related proviruses conserved
region 5 (Fig.
2 and Table
2). The type-specific probes, however,
allowed us to
investigate the detailed distribution of each type
of xenotropic
MLV-related proviruses. For example, using the X-I
probe we could see
that the
Bxv1 provirus fragment comigrated
among three
laboratory strains and one
M. m. molossinus strain
(Fig.
4A,
arrow). Furthermore, although we detected many xenotropic
LTR-reactive
fragments in both
M. m. musculus and
M. m.
molossinus subspecies with the Xltr probe (Fig.
4E and Table
2),
X-II and
X-III are the dominant types in
M. m. musculus and
M. m. molossinus,
respectively (Fig.
4B and C and Table
2).
Laboratory strains
contained roughly equal numbers of the three types
of proviruses,
together accounting for most or all of the Xltr-reactive
fragments.
By contrast, fewer fragments altogether were detected by the
type-specific
probes in
M. m. castaneus,
M. m.
domesticus, and
M. spretus than
by the Xltr probe
(Table
2), indicating that these mice contain
still another type(s) of
xenotropic MLV-related provirus in the
genomes. Note that the P-IV and
P-V proviruses should also be
detected by the Xltr probe (Fig.
3).
Our previous study demonstrated that polytropic MLV-related proviruses
are widely distributed in
M. musculus subspecies and
also
suggested that the proviruses existed in the
Mus germ line
before subspeciation of
M. musculus (
55). To
verify and extend
this observation, we next analyzed the distribution
of the polytropic
MLV-related proviruses using the type-specific
probes (Fig.
5).
The P-I and P-II
specific probes (Pltr and Mltr) detected specific
fragments in
M. musculus and
M. spretus DNAs but not in others
(Fig.
5A
and B). The
M. spretus DNA contained 15 and 2 fragments
specific for the P-I and P-II probes, respectively (Fig.
5A and
B,
lanes f, and Table
2).
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 5.
Distribution of polytropic MLV-related proviruses in
wild mice. Analysis of PvuII-digested mouse DNAs was
performed by hybridization of the DNA in dried agarose gels with the
P-I-specific oligonucleotide probe (Pltr) (A), the P-II-specific
oligonucleotide probe (Mltr) (B), the P-IV-specific oligonucleotide
probe (KT-58) (C), and the P-V-specific oligonucleotide probe (KT-76)
(D). (A and B) Lanes: a, C57BL/6J; b, CASA/Rk (M. m.
castaneus); c, SF/CamEi (M. musculus); d, PERA/Rk
(M. musculus); e, PERC/Ei (M. musculus) (m.mus.);
f, SPRET/Ei (M. spretus) (m.spr.); g, PANCEVO/Ei (M. spicelegus); h, Halbturn (M. spicelegus) (m.spi.); i,
M. cervicolor (m.cer.); j, M. caroli (m.car.); k,
M. cookii (m.coo.). (C and D) a, CZECH II/Ei (M. m.
musculus); b, ZALENDE/Ei (M. m. domesticus) (m.mus.);
c, SPRET/Ei (M. spretus) (m.spr.); d, PANCEVO/Ei (M. spicelegus); e, Halbturn (M. spicelegus) (m.spi.); f,
M. cervicolor (m.cer.); g, M. caroli (m.car.); h,
M. cookii (m.coo.). The approximate positions of molecular
markers are also shown.
|
|
The distribution of the P-IV and P-V proviruses was also examined by
using specific probes. The P-IV provirus-specific probe,
KT-58,
detected two comigrating bands, most likely representing
a single
copy of this type of provirus, in the
M. musculus and
M. spretus species DNAs (Fig.
5C). A similar observation was
made
by using the P-V specific probe, KT-76 (Fig.
5D), with which
fragments
common to
M. spretus and one strain of
M. musculus were also observed.
These observations
suggested that these proviruses were integrated
into the
Mus
germ line before separation between
M. musculus and
M. spretus species. Unfortunately, we could not generate a P-III
provirus-specific probe because it lacks a sequence feature to
distinguish it from the other polytropic MLV-related proviruses.
However, hybridization with the probe, KT-69, that detected all
types
of polytropic MLV-related proviruses except for P-I provirus
implied
that the P-III provirus was found only in
M. musculus subspecies and distributed predominantly in
M. m.
musculus subspecies
(Table
2).
Sequence and phylogenetic analyses of M. spicelegus
endogenous MLV provirus.
Analyses using type-specific probes
suggested the possibility that the X-IV-related provirus might
represent the oldest type of nonecotropic provirus among those
examined, since it was the only one detected in the relatively
distantly related M. spicelegus. Thus, we cloned an
X-IV-reactive fragment from this species in two steps. First, we used a
PCR primer that recognized a conserved MLV group env
sequence, Unienv-3 (Fig. 6A), and an antisense primer, KA-59C,
complementary to the X-IV-specific probe (Fig. 2 and 6A) to amplify and
clone the whole env region. To determine whether this cloned
fragment was the same X-IV-related provirus detected in the analysis
presented Fig. 4D, we hybridized PvuII-digested M. spicelegus DNAs with a specific U3 region probe, KT-61,
designed from the cloned product. As shown in Fig.
6B, KT-61 detected two fragments
identical to those seen with KT-59/60 (Fig. 4D), indicating that the
cloned fragment was indeed derived from the X-IV-related provirus. This
provirus was named hortulanus endogenous MLV (HEMV).
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 6.
Detection of fragments of the M. spicelegus
endogenous provirus (HEMV). (A) The locations of PCR primers and the
oligonucleotide probes used. The approximate positions of the SU and TM
regions of the env gene and the U3 and R regions of the LTR
are indicated. Two hypervariable regions (VRA and VRB) in the SU region
are also shown by boxes. (B) Detection of the HEMV provirus. Analysis
of PvuII-digested mouse DNAs was performed by using the
HEMV-specific oligonucleotide probe KT-61. Lanes: a, CZECH II/Ei
(M. musculus); b, SPRET/Ei (M. spretus); c,
PANCEVO/Ei (M. spicelegus); d, Halbturn (M. spicelegus); e, M. cervicolor; f, M. caroli;
g, M. cookii. The approximate positions of molecular markers
are also shown. (C) Amino acid sequences of the VRA and VRB regions of
the HEMV env gene are aligned with the analogous regions of
several type C retroviruses. Abbreviations and strains of the viruses
included in this alignment are as follows: P-I (MX27) (49);
X-I (NZB) (41); Ampho, amphotropic MLV (4070A)
(42); Eco, ecotropic MLV (Akv) (58); MDEV,
M. dunni endogenous virus (62); GalV, (U20589);
FeLV, feline leukemia virus subgroup A (Glasgow-1) (48).
|
|
To infer the host range of HEMV, we determined the nucleotide sequence
of part of its
env gene. The host range of MLVs is
specified
by two variable regions (VRA and VRB) in the 5' portion
of the surface
(SU) protein (Fig.
6A) (
1). The analysis indicated
that the
amino acid sequences of VRA and VRB of HEMV were quite
different from
those of other type C leukemia viruses (Fig.
6C),
although other parts
of the Env protein were highly conserved.
Most strikingly, HEMV
encoded a very short VRA sequence in the
genome, implying a host
range distinct from that of other known
MLVs.
For the second step, we designed an HEMV-specific sense primer located
in the
env region, HE-8 (Fig.
6A), and performed PCR
amplification with it and the Uniltr-4 antisense primer. The PCR
product was purified and sequenced. By using a specific probe,
the PCR product was confirmed to encode the HEMV U3 region (data
not
shown). The sequence of the HEMV U3 region is shown in Fig.
7. Although this sequence was distinct
from the other proviruses,
it was still closely related to both
ecotropic and nonecotropic
MLV proviruses. A highly conserved 24-bp
region, including the
UCR motif, was present just 3' of the absent
PstI site (Fig.
7A).
HEMV encoded only one region 1 and
the sequence showed a 1-bp
difference from that of the X-IV provirus.
This difference probably
accounts for the relatively weak hybridization
signals seen with
M. spicelegus DNAs by using the KT-59/60
probe (Fig.
4D). Interestingly,
the enhancer regions, including
region 4, were well conserved
among all MLV proviruses (Fig.
7A).
The core-binding and E-box
1 motifs of HEMV were found
in ecotropic and nonecotropic types,
respectively. The DEN region
of HEMV was relatively close to that
of ecotropic proviruses.
Furthermore, HEMV had a complete CAT
box, CCAAT, as was the case for
the X-I and ecotropic proviruses.
Moreover, unlike the U3
sequences of known MLV proviruses (
22),
HEMV lacked both
region 6 and region 6* in the 3' end of U3. Interestingly,
simian
sarcoma virus (SSV) (
13), gibbon ape leukemia virus (GaLV)
(
56), and feline leukemia virus (
48) U3 sequences
also lack
these regions (Fig.
7B).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 7.
Alignment of HEMV U3 sequences. (A) The U3 region of the
HEMV provirus was obtained as described in the text and is aligned with
the corresponding sequences from the X-IV (Mxv11), X-I (NFS-Th-1)
(28), P-I (MX27) (49), and ecotropic (Akv)
(58) proviruses. Dots indicate nucleotide identity. Dashes
indicate absence of a nucleotide. The sequence of an oligonucleotide
probe, KT-59/60, reactive with X-IV proviruses is underlined. Direct
repeats in the enhancer regions found in ecotropic proviruses are not
shown. The PstI recognition site is also shown. (B) Lack of
the region 6 direct repeat in HEMV. The 3' portion of the HEMV U3
sequence is aligned with the analogous regions of the several type C
retroviruses. Abbreviations and strains of the viruses included in this
alignment are as follows: GaLV-SE and GaLV-SF, SEATO and San Francisco
isolates (56); FeLV-A and FeLV-B, feline leukemia virus,
subgroup A (Glasgow-1) and subgroup B (Gardner-Arnstein)
(48); X-I, NFS-Th-1 (28).
|
|
To assess the phylogenetic relationship between HEMV and the other
proviruses, we generated unrooted neighbor-joining phylogenetic
trees for the
env and U3 sequences of the
proviruses presented
here (Fig.
8). In the
env tree (Fig.
8A),
each group of proviruses
was clearly separated from the others.
Interestingly, HEMV was
found to be equidistant from the
ecotropic and nonecotropic proviruses
as well as from the viruses
of various species. The U3 tree also
supports the phylogenetic position
of HEMV and is also supported
by bootstrap analysis (Fig.
8B and C).
These observations support
the possibility that HEMV might resemble a
common ancestor of
recent MLVs.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 8.
Phylogenetic analysis of the nonecotropic U3 proviruses.
Unrooted phylogenetic trees for the env (A) and U3 (B and C)
regions of HEMV and several type C leukemia viruses were estimated by
neighbor joining. Branch lengths are drawn to scale. To illustrate
consistency all bootstrap values obtained with 1,000 replications of
bootstrap sampling are shown. Viruses used for this analysis are as
follows: ecotropic viruses, Akv (58), Moloney
(47), Friend (FE29 strain) (43), Fv4 (M33884),
Cas-Br-E (P08360), and HoMuLV (60); nonecotropic viruses,
P-I (MX27) (49), P-II (MX33) (49), and X-I
(NFS-Th-1) (28); amphotropic viruses, (4070A)
(42), MDEV (62), GaLV (SEATO) (56),
and feline leukemia virus subgroup A (FeLV-A) (Glasgow-1)
(48).
|
|
| |
DISCUSSION |
Genetic variation and distribution of nonecotropic MLV proviruses
in wild mice.
Endogenous nonecotropic MLV proviruses are stable
genetic elements that were fixed recently in the Mus germ
line and are therefore highly polymorphic. Endogenous ecotropic MLV
proviruses are also polymorphic but are neither widely distributed nor
greatly amplified in mice (4, 12), indicative of much more
recent origin. Study of the nonecotropic MLV proviruses should provide
a tool to obtain a better understanding of the association between
retroviruses and their host during their evolutionary history. Here, we
analyzed the extended genetic variation of nonecotropic
endogenous MLV proviruses in wild mice including M. musculus, M. spretus, and M. spicelegus. We report here several new and distinct classes of nonecotropic provirus U3 sequences and their relationships to one
another and to known endogenous proviruses.
The proviruses we studied showed a high degree of sequence similarity
in U3, consistent with a recent evolutionary relationship.
The 5'
portion including the UCR motif, which is highly conserved
among a
large number of type C retroviruses (
22), was also the
most
conserved among the wild-mouse proviruses. This motif has
been reported
to function as a negative regulatory element in
several murine type C
viruses (
17). Other regions of U3 are
less well conserved
and allowed us to group the proviruses into
a number of subtypes, using
polymorphisms arising from duplication,
deletion, insertion, or point
mutation. A schematic view of the
relationship among the various groups
is shown in Fig.
9, in the
form of a
possible scheme for their evolution. For example, type
X-I proviruses
contain distinctive enhancer-promoter sequences
in the U3 region (Fig.
2). All X-I proviruses include the consensus
MLV core-enhancer binding
motif and the factor binding sites in
region 4 but not the direct
repeats of region 4*, and almost all
contain a unique set of factor
binding sequences in the DEN region.
Furthermore, the
promoter-associated CAT box of these proviruses
was a perfect match to
the consensus.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 9.
A possible evolutionary scheme relating nonecotropic MLV
U3 regions. Schematic representations of the U3 structures are shown.
Locations of the UCR and CAT box are also shown. Corresponding clones
are shown on the right of the structures. Clone pRFM17 was described
previously (8). Note that this scheme is parsimonious with
respect to insertions and deletions. Discrepancies relative to the tree
shown in Fig. 8 probably stem from these events having occurred
multiple times.
|
|
Although we have not studied the functions of the enhancer-promoter
sequences in the wild-mouse proviruses, the U3 regions
of retroviruses,
in general, are intimately involved in viral
replication and
pathogenicity (
24,
33,
35,
36,
44,
53,
57,
64). Among all
known nonecotropic proviruses, only the
X-I group includes proviruses
capable of yielding infectious virus
(
28,
35,
41). In this
regard, lack of region 4* of the X-I
proviruses could be critical.
Duplication of core-enhancer regions
of MLVs is associated with
increases in transcriptional activity
(
9,
10,
35,
38,
57),
and strong enhancer activity in
retroviruses often results in increased
pathogenicity for the
host (
33,
35,
44,
53,
57). Indeed,
leukemogenicity of
MCF viruses in certain mice is associated with and
appears to
be dependent on the acquisition (by recombination) of an
X-I-type
LTR followed by duplication of region 4 enhancer sequences
(
53).
In fact, MCF viruses harboring such repeated sequences
have not
been detected as endogenous elements in the mice
(
55). Considering
these observations, it is likely that X-I
proviruses containing
duplicated enhancer sequences could not remain in
the mouse genome
as endogenous elements on account of their strong
enhancer
activity.
The X-II and X-III proviruses were virtually identical to each other,
and X-III was most likely generated by a deletion in
the X-II provirus
(Fig.
2 and
9). Members of these two groups
contained several unique
point differences or deletions in their
U3 sequences that differentiate
them from others. Both X-II and
X-III proviruses have been isolated in
previous studies, although
the sequence characteristics were not
examined. Two provirus clones
harboring the X-II-type U3 structure,
pGP24 and pGP68 (
46),
were cloned from a cDNA library from
the liver of NZB mice and
found to encode the
env
glycoprotein (gp70) found in serum and
expressed as an acute-phase
protein in mice. Sequences related
to these clones are highly expressed
in the liver and kidney tissues
of NZB mice injected with
lipopolysaccharide (
46). Furthermore,
two independent
isolates, MRV (
11) and EDV (
31), which are
related to the murine AIDS (MAIDS) virus, were identified as proviruses
containing the X-II-type U3 in their genomes. These two MAIDS-related
viruses are endogenous in laboratory strains and have donated
the
gag sequences characteristic of MAIDS viruses to the related
exogenous viruses by recombination (
11,
31). The X-III
proviruses
have also been cloned before. Insertion of
Xmv28,
an X-III provirus,
into the mouse genome is associated with the
rd mutation (
6).
The distribution of the proviruses also has important implications for
the evolutionary relationship of the xenotropic MLVs.
First, most of
the xenotropic relatives were found only in
M. musculus
subspecies (Fig.
4 and Table
2). In particular,
M. m. musculus and
M. m. molossinus were the predominant
carriers of
the X-I, X-II, and X-III proviruses, indicating that they
entered
the mouse germ line after separation between
M. musculus and
M. spretus, probably around the time of
subspeciation between
M. m. musculus and
M. m.
domesticus. Despite their presence, the
dissimilarities of the
distributions and numbers of the proviruses
among the subspecies imply
that a variety of amplification and
germ line integration events
occurred after separation of the
subspecies. Furthermore, the
type-specific probes revealed introgression
of some proviruses involved
in pathogenicity or mutation into
certain laboratory mice. The
inbred strains could have inherited
the
Bxv1 MCF-related
provirus from the
M. m. molossinus subspecies
(Fig.
4A). The MAIDS-related proviruses could have been generated
after
subspeciation of
M. musculus and then acquired in laboratory
strains from either
M. m. musculus or
M. m.
molossinus (Fig.
4B).
Two mutation-associated proviruses,
Xmv10 and
Xmv28 (
6,
61),
were probably
inherited from the
M. m. molossinus subspecies (Fig.
4C).
Further study of the distribution among mice of provirus
integrated at specific sites will be required to determine
the
relationship between integration and speciation
events.
Among xenotropic MLV-related proviruses, the X-IV type showed unique
genetic features and a unique distribution pattern in
the mice. Despite
a xenotropic MLV-related U3 structure, the X-IV
proviruses shared some
common features with polytropic MLV-related
proviruses, including
identical region 1 and CAT box sequences.
Furthermore, some X-IV
proviruses and Poly/Xeno recombinant proviruses
from
M. m.
musculus and
M. m. domesticus contain polytropic
virus-type
env sequences (
55). Sequence analysis
of a clone from our genomic
library, Mxv11, indicated that the SU
region of the provirus was
more similar to that of polytropic viruses
than to that of xenotropic
viruses, although it contained a large
deletion (data not shown).
These observations imply that this type of
provirus could be related
to a recent common ancestor of the
nonecotropic proviruses. It
could be necessary to investigate larger
numbers of samples from
M. spretus or the related species to
verify this
hypothesis.
Polytropic MLV-related proviruses showed less diversity than xenotropic
MLV-related proviruses in the wild mice (Fig.
3).
Sequence analysis
demonstrated that proviruses analogous to polytropic
(type P-I) and
modified polytropic (P-II) proviruses in laboratory
strains
(
49) were also present and widely distributed in
M. spretus. This result was also confirmed by unblotting (Fig.
5 and
Table
2). Among the polytropic MLV-related proviruses, the
predominant
proviruses were of the P-I and P-II types, while P-IV
and P-V were not
widespread, although some proviruses yielded
comigrating bands from
DNAs from two distinct species,
M. musculus and
M. spretus (Fig.
5C and D). This result implies that polytropic
MLV-related proviruses were integrated into the
Mus germ
line
before separation between these species, but that only the P-I
and
P-II proviruses were able to spread within the
Mus genomes.
Furthermore, the sequence analysis revealed that both the P-I
and P-II
proviruses contain an internal deletion in the 5' portion
of the 190-bp
unique insertion (Fig.
3), while the P-III, P-IV
and P-V proviruses
contained intact forms of the inserted sequence
in their genomes. This
observation suggests that the P-I and P-II
proviruses were derived from
other polytropic MLV-related proviruses
with an intact 190-bp
insertion. It is likely that P-II was generated
from the P-III provirus
by simple deletion in the inserted sequence
(see
below).
One of the interesting findings of this study was obtained from
analysis of the provirus from
M. spicelegus, HEMV. Although
originally detected with the type X-IV probe, the U3 region of
this
provirus does not belong with either the xenotropic or the
polytropic
group. HEMV junction fragments did comigrate in two
different strains
of
M. spicelegus, indicating that it is indeed
endogenous in
these mice. In a previous study, a distantly related
pathogenic
ecotropic virus (Hortulanus MLV or HoMLV) was isolated
from
M. spicelegus species (
59,
60). It is believed,
however,
that HoMLV is exogenous and was acquired from Asian wild mice
(
59,
60). The sequence and phylogenetic analyses of HEMV
clearly
demonstrate its close relationship to MLVs, but it is
equidistant
from ecotropic and nonecotropic provirus groups (Fig.
7B
and
8).
These results suggest that HEMV is likely to be related to the
common ancestor of MLVs. Despite the ancestral phylogenetic position
of
HEMV, however, the provirus is not widespread in wild mice
(Fig.
6B).
This result implies a more recent spread by infection
of the provirus
rather than inheritance. Furthermore, the phylogenetic
position of HEMV
was also found to be equidistant from simian
leukemia viruses
(GaLV and SSV) and MLVs. Indeed, the sequence
analysis also revealed
that the 3' end of its U3 region is more
closely related to the simian
viruses than to the murine proviruses
(Fig.
7B). It is now
believed that GaLV and SSV originated subsequent
to a transspecies
infection of primates by xenotropic viruses
of murine origin derived
from
M. caroli,
M. cervicolor, or other
related
mouse species (
2,
34). Although sequence analysis
of the
xenotropic viruses from
M. caroli and
M. cervicolor has
not been carried out and the HEMV sequence was not
detected in
these species (Fig.
6B), there is a possibility that HEMV
is the
origin of the GaLV/SSV group in primates. Moreover, an
endogenous
virus (MDEV) that shows a unique host range has recently
been
isolated from
M. dunni (
5). The
sequences of the vrA and vrB
regions of both MDEV (
62) and
HEMV are consistent with a host
range different from that of all
other known viruses of this genus
(Fig.
6C). However, they are
also quite different from each other
and occupy distinct
phylogenetic positions (Fig.
8A).
Possible evolutionary relationships among MLVs in wild mice.
The unique structural features and detailed distribution of the
nonecotropic provirus U3 regions raise several interesting possibilities regarding their evolutionary relationships. In the scheme
proposed in Fig. 9, HEMV could represent the progenitor of the
LTRs found in other nonecotropic proviruses. The HEMV U3 sequence could have evolved into the nonecotropic-type U3 and given rise to proviruses like the X-IV type following several mutations, including insertion of regions 6 and 6* and a number of
duplication and recombination events. The X-IV sequences could generate
P-IV and P-V proviruses by insertion of the 190-bp sequence and
deletion of region 6*. The polytropic virus-like env
sequence of the X-IV proviruses supports this evolutionary step. The
P-IV and P-V proviruses seem to be ancestral forms of the polytropic MLV-related proviruses. The P-III sequence could have been derived from
P-IV and P-V following duplication of region 1, and it could have given
rise to P-I and P-II proviruses by deletions including the 5' side of
the 190-bp insertion. The apparent presence of type P-IV and P-V
proviruses in both M. musculus and M. spretus suggests that the separation of these viruses occurred before the
separation of M. musculus and M. spretus about 5 million years ago. Type X-I proviruses could have been derived from
type X-IV proviruses by several mutations. The X-II and X-III
proviruses could have been generated by duplication of region 1 from
the X-I proviruses or recombination with polytropic MLV-related
proviruses. These events could have occurred after the separation
of M. musculus and M. spretus.
In order to accomplish these evolutionary steps, it would be necessary
for the proviruses to have extended periods of replication
as viruses
and involve numerous mutational and recombinational
events. Both the
nature of the mutational events (duplications,
insertions, and
deletions, particularly involving enhancer regions)
and their rapid
accumulation over short evolutionary periods are
inconsistent with
mutations occurring during residence in the
mouse germ line. These
events are consistent with those commonly
found in the generation of
the recombinant MCF and MAIDS viruses
derived in part from the X-I and
X-III proviruses, respectively.
Furthermore, a mosaic provirus, which
has the type X-I enhancer-DEN
region on the X-II- or X-III-based
sequence, was found in a strain
of
M. m. molossinus,
Mcv6 (Fig.
9), directly implicating recombination
during virus
replication.
It is important to keep in mind that the scheme shown in Fig.
9 is the
simplest, involving the smallest number of insertion,
duplication, and
deletion events. It shares many features with
the point mutation-based
tree shown in Fig.
8, but the two patterns
are not perfectly
concordant, particularly in the polytropic lineage.
This discordance
implies that some events may have occurred more
than once during the
evolution of these viruses. Given the high
frequency of recombination
and duplication events observed during
infection of a single animal
(
53), it is likely that some of
the duplication and
deletion events have occurred multiple times
during the evolution
of these
viruses.
Figure
10 shows a schematic
representation of the coevolution of MLV proviruses and the wild-mouse
species we included in this
study. The observation that the
ancestral-type proviruses are
not widespread in mice implies that these
proviruses only recently
expanded into these mice by infection rather
than inheritance.
Thus, rather than serving as either a static
repository for ancient
viruses or a reservoir from which exogenous
viruses appear from
time to time, endogenous proviruses should be
viewed as a snapshot
of a complex dynamic process involving long
periods of replication
as viruses, during which recombination and
mutation events accumulate,
and relatively recent germ line insertion.
These proviruses could
provide new genetic markers for the strain
identification or evolutionary
study of both MLVs and their murine
host. The approach taken in
this study will serve as the basis for
further examination of
the evolution and involvement of retroviral
sequences in tumorigenicity
in the murine host.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 10.
Coevolution of MLVs and wild mice. A schematic
phylogenetic relationship of wild-mouse species is shown. Possible
periods of provirus integration are represented by arrows. The bar
under the tree indicates the approximate time scale. MYA, million years
ago.
|
|
| |
ACKNOWLEDGMENTS |
We are grateful to Jonathan Stoye and Christopher Tipper for
helpful comments, to Mary Bostic-Fitzgerald for preparing the manuscript, and to Christine Kozak for the generous gift of wild-mouse DNAs.
This work was supported by National Cancer Institute award R35CA44385
to J.M.C. and a Leukemia Society of America Special Fellowship to K.T.
J.M.C. was a research professor of the American Cancer Society.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Microbiology, Tufts University, School of
Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6528. Fax: (617) 636-4086. E-mail:
jcoffin_par{at}opal.tufts.edu.
| |
REFERENCES |
| 1.
|
Battini, J.-L.,
J. M. Heard, and O. Danos.
1992.
Receptor choice determinants in the envelope glycoprotein of amphotropic, xenotropic, and polytropic murine leukemia viruses.
J. Virol.
66:1468-1475[Abstract/Free Full Text].
|
| 2.
|
Benveniste, R. E.,
R. Callahan,
C. J. Sherr,
V. Chapman, and G. J. Todaro.
1977.
Two distinct endogenous type C viruses isolated from the Asian rodent Mus cervicolor: conservation of virogene sequences in related rodent species.
J. Virol.
21:849-862[Abstract/Free Full Text].
|
| 3.
|
Blatt, C.,
K. Mileham,
M. Haas,
M. N. Nesbitt,
M. E. Harper, and M. I. Simon.
1983.
Chromosomal mapping of the mink cell focus-inducing and xenotropic env gene family in the mouse.
Proc. Natl. Acad. Sci. USA
80:6298-6302[Abstract/Free Full Text].
|
| 4.
|
Boeke, J. D., and J. P. Stoye.
1997.
Retrotransposons, endogenous retroviruses, and the evolution of retroelements, p. 343-435.
In
J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 5.
|
Bonham, L.,
G. Wolgamot, and A. D. Miller.
1997.
Molecular cloning of Mus dunni endogenous virus: an unusual retrovirus in a new murine viral interference group with a wide host range.
J. Virol.
71:4663-4670[Abstract].
|
| 6.
|
Bowes, C.,
T. Li,
W. N. Frankel,
M. Danciger,
J. M. Coffin,
M. L. Applebury, and D. B. Farber.
1993.
Localization of a retroviral element within the rd gene coding for the b subunit of cGMP-phosphodiesterase.
Proc. Natl. Acad. Sci. USA
90:2955-2959[Abstract/Free Full Text].
|
| 7.
|
Ch'ang, L.-Y.,
W. K. Yank,
F. E. Myer,
C. K. Koh, and L. R. Boone.
1989.
Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome.
Virology
168:245-255[Medline].
|
| 8.
|
Ch'ang, L.-Y.,
W. K. Yang,
F. E. Myer, and D.-M. Yang.
1989.
Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.
J. Virol.
63:2746-2757[Abstract/Free Full Text].
|
| 9.
|
Chattopadhyay, S. K.,
H. C. I. Morse,
M. Makino,
S. K. Ruscetti, and J. W. Hartley.
1989.
Defective virus is associated with induction of murine retrovirus-induced immunodeficiency syndrome.
Proc. Natl. Acad. Sci. USA
86:3862-3866[Abstract/Free Full Text].
|
| 10.
|
Chen, H., and F. K. Yoshimura.
1994.
Identification of a region of a murine leukemia virus long terminal repeat with novel transcriptional regulatory activities.
J. Virol.
68:3308-3316[Abstract/Free Full Text].
|
| 11.
|
Cho, B. C.,
J. J. Shaughnessy,
D. A. Largaespada,
H. G. Bedigian,
A. M. Buchberg,
N. A. Jenkins, and N. G. Copeland.
1995.
Frequent disruption of the Nf1 gene by a novel murine AIDS virus-related provirus in BXH-2 murine myeloid lymphomas.
J. Virol.
69:7138-7146[Abstract].
|
| 12.
|
Coffin, J. M.,
J. P. Stoye, and W. N. Frankel.
1989.
Genetics of endogenous murine leukemia viruses.
Ann. N. Y. Acad. Sci.
567:39-49[Medline].
|
| 13.
|
Devare, S. G.,
E. P. Reddy,
J. D. Law,
K. C. Robbins, and S. A. Aaronson.
1983.
Nucleotide sequence of the simian sarcoma virus genome: demonstration that its acquired cellular sequences encode the transforming gene product p28sis.
Proc. Natl. Acad. Sci. USA
80:731-735[Abstract/Free Full Text].
|
| 14.
|
Devereux, J.,
P. Haeberli, and O. Smithies.
1984.
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res.
12:387-395.
|
| 15.
|
Felsenstein, J.
1985.
Confidence limits on phylogenies: an approach using the bootstrap.
Evolution
39:783-791.
|
| 16.
|
Felsenstein, J.
1995.
PHYLIP: phylogeny inference package, version 3.5.
University of Washington, Seattle.
|
| 17.
|
Flanagan, J. R.,
A. M. Krieg,
E. E. Max, and A. S. Khan.
1989.
Negative control region at the 5' end of murine leukemia virus long terminal repeats.
Mol. Cell. Biol.
9:739-746[Abstract/Free Full Text].
|
| 18.
|
Frankel, W. N., and J. M. Coffin.
1994.
Endogenous nonecotropic proviruses mapped with oligonucleotide probes from the long terminal repeat region.
Mamm. Genome
5:275-281[Medline].
|
| 19.
|
Frankel, W. N.,
J. P. Stoye,
B. A. Taylor, and J. M. Coffin.
1989.
Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1.
J. Virol.
63:1763-1774[Abstract/Free Full Text].
|
| 20.
|
Frankel, W. N.,
J. P. Stoye,
B. A. Taylor, and J. M. Coffin.
1989.
Genetic identification of endogenous polytropic proviruses by using recombinant inbred mice.
J. Virol.
63:3810-3821[Abstract/Free Full Text].
|
| 21.
|
Frankel, W. N.,
J. P. Stoye,
B. A. Taylor, and J. M. Coffin.
1990.
A linkage map of endogenous murine leukemia proviruses.
Genetics
124:221-236[Abstract].
|
| 22.
|
Golemis, E. A.,
N. A. Speck, and N. Hopkins.
1990.
Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design.
J. Virol.
64:534-542[Abstract/Free Full Text].
|
| 23.
|
Hoggan, M. D.,
R. R. O'Neill, and C. A. Kozak.
1986.
Nonecotropic murine leukemia viruses in BALB/c and NFS/N mice: characterization of the BALB/c Bxv-1 provirus and the single NFS endogenous xenotrope.
J. Virol.
60:980-986[Abstract/Free Full Text].
|
| 24.
|
Holland, C. A.,
C. Y. Thomas,
S. K. Chattopadhyay,
C. Koehne, and P. V. O'Donnell.
1989.
Influence of enhancer sequences on thymotropism and leukemogenicity of mink cell focus-forming viruses.
J. Virol.
63:1284-1292[Abstract/Free Full Text].
|
| 25.
|
Inaguma, Y.,
N. Miyashita,
K. Moriwaki,
W. C. Huai,
J. Mei-Lei,
H. Zinqiao, and H. Ikeda.
1991.
Acquisition of two endogenous ecotropic murine leukemia viruses in distinct Asian wild mouse populations.
J. Virol.
65:1796-1802[Abstract/Free Full Text].
|
| 26.
|
Jenkins, N. A.,
N. G. Copeland,
B. A. Taylor, and B. K. Lee.
1982.
Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus.
J. Virol.
43:26-36[Abstract/Free Full Text].
|
| 27.
|
Khan, A. S.
1984.
Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.
J. Virol.
50:864-871[Abstract/Free Full Text].
|
| 28.
|
Khan, A. S., and M. A. Martin.
1983.
Endogenous murine leukemia proviral long terminal repeats contain a unique 190-base-pair insert.
Proc. Natl. Acad. Sci. USA
80:2699-2703[Abstract/Free Full Text].
|
| 29.
|
Kimura, M.
1980.
A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.
J. Mol. Evol.
16:111-120[Medline].
|
| 30.
|
Kozak, C. A., and R. R. O'Neill.
1987.
Diverse wild mouse origins of xenotropic, mink-cell focus-forming, and two types of ecotropic proviral genes.
J. Virol.
61:3082-3088[Abstract/Free Full Text].
|
| 31.
|
Kubo, Y.,
Y. Nakagawa,
K. Kakimi,
H. Matsui,
K. Higo,
L. Wang,
H. Kobayashi,
T. Hirama, and A. Ishimoto.
1994.
Molecular cloning and characterization of a murine AIDS virus-related endogenous transcript expressed in C57BL/6 mice.
J. Gen. Virol.
78:881-888.
|
| 32.
|
Lamont, C.,
P. Culp,
R. L. Talbott,
T. R. Phillips,
R. J. Trauger,
W. N. Frankel,
M. C. Wilson,
J. M. Coffin, and J. H. Elder.
1991.
Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line.
J. Virol.
65:4619-4628[Abstract/Free Full Text].
|
| 33.
|
Lenz, J.,
D. Celander,
R. L. Crowther,
R. Patarca,
D. W. Perkins, and W. A. Haseltine.
1984.
Determination of the leukemogenicity of a murine retrovirus by sequences within the long terminal repeat.
Nature
308:467-470[Medline].
|
| 34.
|
Lieber, M. M.,
C. J. Sherr,
G. J. Todaro,
R. E. Benveniste,
R. Callahan, and H. G. Coon.
1975.
Isolation from the Asian mouse Mus caroli of an endogenous type C virus related to infectious primate type C viruses.
Proc. Natl. Acad. Sci. USA
72:2315-2319[Abstract/Free Full Text].
|
| 35.
|
Massey, A. C.,
M. A. Coppola, and C. Y. Thomas.
1990.
Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.
J. Virol.
64:5491-5499[Abstract/Free Full Text].
|
| 36.
|
Massey, A. C.,
S. S. Lawrenz,
D. J. Innes, and C. Y. Thomas.
1994.
Origins of enhancer sequences of recombinant murine leukemia viruses from spontaneous B- and T-cell lymphomas of CWD mice.
J. Virol.
68:3773-3783[Abstract/Free Full Text].
|
| 37.
|
Meruelo, D.,
A. Rossomando,
M. Offer,
J. Buxbaum, and A. Pellicer.
1983.
Association of endogenous viral loci with genes encoding murine histocompatibility and lymphocyte differentiation antigens.
Proc. Natl. Acad. Sci. USA
80:5032-5036[Abstract/Free Full Text].
|
| 38.
|
Morrison, H. L.,
B. Soni, and J. Lenz.
1995.
Long terminal repeat enhancer core sequences in proviruses adjacent to c-myc in T-cell lymphomas induced by a murine retrovirus.
J. Virol.
69:446-455[Abstract].
|
| 39.
|
Needleman, S. B., and C. D. Wunsch.
1970.
A general method applicable to the search for similarities in the amino acid sequence of two proteins.
J. Mol. Biol.
48:443-453[Medline].
|
| 40.
|
O'Brien, S. J.,
J. L. Moore,
M. A. Martin, and J. E. Womack.
1982.
Evidence for the horizontal acquisition of murine AKR virogenes by recent horizontal infection of the germ line.
J. Exp. Med.
155:1120-1123[Abstract/Free Full Text].
|
| 41.
|
O'Neill, R. R.,
C. E. Buckler,
T. S. Theodore,
M. A. Martin, and R. Repaske.
1985.
Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus.
J. Virol.
53:100-106[Abstract/Free Full Text].
|
| 42.
|
Ott, D.,
R. Friedrich, and A. Rein.
1990.
Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses.
J. Virol.
64:757-766[Abstract/Free Full Text].
|
| 43.
|
Perryman, S.,
J. Nishio, and B. Chesebro.
1991.
Complete nucleotide sequence of Friend murine leukemia virus, strain FB29.
Nucleic Acids Res.
19:6950[Free Full Text].
|
| 44.
|
Rosen, C. A.,
W. A. Haseltine,
J. Lenz,
R. Ruprecht, and M. W. Cloyd.
1985.
Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences.
J. Virol.
55:862-866[Abstract/Free Full Text].
|
| 45.
|
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 46.
|
Shigemoto, K.,
S. Kubo,
Y. Itoh,
G. Tate,
S. Handa, and N. Maruyama.
1992.
Expression and structure of serum gp70 as an acute phase protein in NZB mice.
Mol. Immunol.
29:573-582[Medline].
|
| 47.
|
Shinnick, T. M.,
R. A. Lerner, and J. G. Sutcliffe.
1981.
Nucleotide sequence of Moloney murine leukaemia virus.
Nature
293:543-548[Medline].
|
| 48.
|
Stewart, M. A.,
M. Warnock,
A. Wheeler,
N. Wilkie,
J. I. Mullins,
D. E. Onions, and J. C. Neil.
1986.
Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses.
J. Virol.
58:825-834[Abstract/Free Full Text].
|
| 49.
|
Stoye, J. P., and J. M. Coffin.
1987.
The four classes of endogenous murine leukemia virus: structural relationships and potential for recombination.
J. Virol.
61:2659-2669[Abstract/Free Full Text].
|
| 50.
|
Stoye, J. P., and J. M. Coffin.
1988.
Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes.
J. Virol.
62:168-175[Abstract/Free Full Text].
|
| 51.
|
Stoye, J. P.,
W. N. Frankel, and J. M. Coffin.
1991.
DNA hybridization in dried gels with fragmented probes: an improvement over blotting techniques.
Technique
3:123-128.
|
| 52.
|
Stoye, J. P.,
N. Kaushik,
S. Jeremiah, and S. Best.
1995.
Genetic map of the region surrounding the retrovirus restriction locus, Fv1, on mouse chromosome 4.
Mamm. Genome
6:31-36[Medline].
|
| 53.
|
Stoye, J. P.,
C. Moroni, and J. Coffin.
1991.
Virological events leading to spontaneous AKR thymomas.
J. Virol.
65:1273-1285[Abstract/Free Full Text].
|
| 54.
|
Taylor, B. A.
1978.
Recombinant inbred strains: use in gene mapping, p. 423-438.
In
H. C. Morse III (ed.), Origins of inbred mice. Academic Press, New York, N.Y.
|
| 55.
|
Tomonaga, K., and J. M. Coffin.
1998.
Structure and distribution of endogenous nonecotropic murine leukemia viruses in wild mice.
J. Virol.
72:8289-8300[Abstract/Free Full Text].
|
| 56.
|
Trainor, C. D.,
M. L. Scott,
S. F. Josephs,
K. E. Fry, and M. J. Reitz.
1984.
Nucleotide sequence of the large terminal repeat of two different strains of gibbon ape leukemia virus.
Virology
137:201-205[Medline].
|
| 57.
|
Tupper, J. C.,
H. Chen,
E. F. Hays,
G. C. Bristol, and F. K. Yoshimura.
1992.
Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer.
J. Virol.
66:7080-7088[Abstract/Free Full Text].
|
| 58.
|
Van Beveren, C.,
E. Rands,
S. K. Chattopadhyay,
D. R. Lowy, and I. M. Verma.
1982.
Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.
J. Virol.
41:542-556[Abstract/Free Full Text].
|
| 59.
|
Voytek, P., and C. Kozak.
1988.
HoMuLV: a novel pathogenic ecotropic virus isolated from the European mouse, Mus hortulanus.
Virology
165:469-475[Medline].
|
| 60.
|
Voytek, P., and C. A. Kozak.
1989.
Nucleotide sequence and mode of transmission of the wild mouse ecotropic virus, HoMuLV.
Virology
173:58-67[Medline].
|
| 61.
|
Winkes, B. M.,
M. M. Ollmann, and G. S. Barsh.
1994.
Association of Xmv-10 and the non-agouti (a) mutation explained by close linkage instead of causality.
Mamm. Genome
5:3-10[Medline].
|
| 62.
|
Wolgamot, G.,
L. Bonham, and A. D. Miller.
1998.
Sequence analysis of Mus dunni endogenous virus reveals a hybrid VL30/gibbon ape leukemia virus-like structure and a distinct envelope.
J. Virol.
72:7459-7466[Abstract/Free Full Text].
|
| 63.
|
Yonekawa, H.,
O. Gotoh,
Y. Tagashira,
Y. Matsushima,
L. I. Shi,
W. S. Cho,
N. Miyashita, and K. Moriwaki.
1986.
A hybrid origin of Japanese mice "Mus musculus molossinus".
Curr. Top. Microbiol. Immunol.
127:62-67[Medline].
|
| 64.
|
Yoshimura, F. K.,
B. Davison, and K. Chaffin.
1985.
Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner.
Mol. Cell. Biol.
5:2832-2835[Abstract/Free Full Text].
|
Journal of Virology, May 1999, p. 4327-4340, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Yoshinobu, K., Baudino, L., Santiago-Raber, M.-L., Morito, N., Dunand-Sauthier, I., Morley, B. J., Evans, L. H., Izui, S.
(2009). Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous Modified Polytropic Retrovirus by the Sgp3 Locus of Lupus-Prone Mice. J. Immunol.
182: 8094-8103
[Abstract]
[Full Text]
-
Baudino, L., Yoshinobu, K., Morito, N., Kikuchi, S., Fossati-Jimack, L., Morley, B. J., Vyse, T. J., Hirose, S., Jorgensen, T. N., Tucker, R. M., Roark, C. L., Kotzin, B. L., Evans, L. H., Izui, S.
(2008). Dissection of Genetic Mechanisms Governing the Expression of Serum Retroviral gp70 Implicated in Murine Lupus Nephritis. J. Immunol.
181: 2846-2854
[Abstract]
[Full Text]
-
Jern, P., Sperber, G. O., Blomberg, J.
(2006). Divergent Patterns of Recent Retroviral Integrations in the Human and Chimpanzee Genomes: Probable Transmissions between Other Primates and Chimpanzees. J. Virol.
80: 1367-1375
[Abstract]
[Full Text]
-
Tipper, C. H., Bencsics, C. E., Coffin, J. M.
(2005). Characterization of Hortulanus Endogenous Murine Leukemia Virus, an Endogenous Provirus That Encodes an Infectious Murine Leukemia Virus of a Novel Subgroup. J. Virol.
79: 8316-8329
[Abstract]
[Full Text]
-
Jung, Y. T., Wu, T., Kozak, C. A.
(2003). Characterization of Recombinant Nonecotropic Murine Leukemia Viruses from the Wild Mouse Species Mus spretus. J. Virol.
77: 12773-12781
[Abstract]
[Full Text]
-
Evans, L. H., Lavignon, M., Taylor, M., Alamgir, A. S. M.
(2003). Antigenic Subclasses of Polytropic Murine Leukemia Virus (MLV) Isolates Reflect Three Distinct Groups of Endogenous Polytropic MLV-Related Sequences in NFS/N Mice. J. Virol.
77: 10327-10338
[Abstract]
[Full Text]
-
Hein, S., Prassolov, V., Zhang, Y., Ivanov, D., Lohler, J., Ross, S. R., Stocking, C.
(2003). Sodium-Dependent myo-Inositol Transporter 1 Is a Cellular Receptor for Mus cervicolor M813 Murine Leukemia Virus. J. Virol.
77: 5926-5932
[Abstract]
[Full Text]
-
Jung, Y. T., Kozak, C. A.
(2003). Generation of Novel Syncytium-Inducing and Host Range Variants of Ecotropic Moloney Murine Leukemia Virus in Mus spicilegus. J. Virol.
77: 5065-5072
[Abstract]
[Full Text]
-
Ikeda, H., Kato, K., Kitani, H., Suzuki, T., Yoshida, T., Inaguma, Y., Yamamoto, N., Suh, J.-G., Hyun, B.-H., Yamagata, T., Namikawa, T., Tomita, T.
(2001). Virological Properties and Nucleotide Sequences of Cas-E-Type Endogenous Ecotropic Murine Leukemia Viruses in South Asian Wild Mice, Mus musculus castaneus. J. Virol.
75: 5049-5058
[Abstract]
[Full Text]
-
Prassolov, V., Hein, S., Ziegler, M., Ivanov, D., Münk, C., Löhler, J., Stocking, C.
(2001). Mus cervicolor Murine Leukemia Virus Isolate M813 Belongs to a Unique Receptor Interference Group. J. Virol.
75: 4490-4498
[Abstract]
[Full Text]
-
Dang, Q., Hu, W.-S.
(2001). Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication. J. Virol.
75: 809-820
[Abstract]
[Full Text]
Top
Abstract
Introduction
