JOURNAL OF VIROLOGY, Mar. 2009, p. 2429–2435
Vol. 83, No. 6
Mobilization of Endogenous Retroviruses in Mice after Infection with
an Exogenous Retrovirus?
Leonard H. Evans,1* A. S. M. Alamgir,1Nick Owens,1Nick Weber,1Kimmo Virtaneva,1
Kent Barbian,2Amenah Babar,2Frank Malik,1and Kyle Rosenke1
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
Hamilton, Montana 59840,1and Research Technologies Branch, RML Research Technologies Section, Genomics Unit,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 598402
Received 12 September 2008/Accepted 22 December 2008
Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during
evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral
genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous
polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these
recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from
an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have
not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recom-
bination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript
from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is
possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus,
infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in
the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral
virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact
endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are
integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The
mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major
interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the
pathogenicity of retroviral infections.
Although the existence of endogenous retroviruses in the
genomes of vertebrate species, including mice and humans, has
been known for many years, it was not until the sequencing of
the human and mouse genomes that the extent to which we
harbor endogenous retroviral sequences was fully appreciated.
It has been estimated that our germ line has incurred about
40,000 infections by retroviruses over the course of evolution
and that retroviral sequences account for 8 to 10% of our total
DNA (20, 38). Most of these retrovirus elements are defective,
and many consist only of solo long terminal repeats (LTRs)
generated by recombination between the two LTRs generated
during the integration process. Others that retain coding se-
quences are often transcriptionally silent; however, some of the
sequences are expressed in a very controlled manner through-
out the lifetime of the host and appear to be modulated under
various physiological and pathological circumstances (4, 5, 23,
26, 29). There are an increasing number of studies indicating
that many of these elements have been utilized for a number of
physiological processes. These include processes at the
genomic level, such as transcriptional control of several genes
(3, 7, 22, 25), at the transcript level, by interaction of viral
RNAs with proteins (35), and at the protein level, such as the
role of endogenous retrovirus envelope proteins in the fusion
of placental trophoblasts (6, 13, 33). The observations that
endogenous retrovirus sequences are expressed during the life-
times of animals may reflect requirements for the transcription
and translation of some of the endogenous retroviruses, even
though the expression of these viruses may have deleterious
consequences. Thus, some restriction factors likely reflect
mechanisms evolved by the host to control endogenous viruses
that are obliged to be transcribed due to a physiological role.
Among the most extensively studied groups of endogenous
retroviruses are the endogenous polytropic retroviruses of
mice (11, 12, 15, 17, 19, 36). These viruses, like some human
retroviruses, are expressed in a tightly controlled fashion dur-
ing the lifetime of the host and have not been found to produce
infectious viruses, even though some of them possess intact env
genes and are transcribed (1, 9, 28, 31, 36). They do, however,
interact with exogenous viruses that have infected the mouse.
Upon infection, the exogenous viruses recombine with the env
gene sequences of the endogenous polytropic MuLVs to gen-
erate host range variants that utilize a different cell surface
receptor for infectious entry (11, 15, 16, 17, 32, 37). The gen-
eration of such recombinant viruses is instrumental in the
induction of disease by a number of exogenous retroviruses.
Recombination between the exogenous and endogenous ret-
rovirus genomes requires transcription of a complete endoge-
nous provirus to an RNA strand that is copackaged with an
exogenous murine leukemia virus (MuLV) transcript as a het-
erodimeric virion RNA. Upon subsequent infection, the het-
* Corresponding author. Mailing address: Laboratory of Persistent
Viral Diseases, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, Hamilton, MT 59840. Phone: (406)
363-9374. Fax: (406) 363-9286. E-mail: email@example.com.
?Published ahead of print on 30 December 2008.
erodimer can undergo recombination during reverse transcrip-
tion (RT) (16, 37). Although the endogenous polytropic
proviruses are transcribed, replication of the endogenous poly-
tropic viruses in the absence of recombination has not been
observed. This may, in many cases, reflect defects, such as
point mutations or deletions, in the endogenous viral genome
but may also be influenced by the activities of various restric-
tion factors (14, 27). The fact that exogenous MuLVs are
capable of replicating in mice indicates that they have evolved
mechanisms to circumvent the activities of at least some of the
restriction factors. Thus, exogenous retroviruses might facili-
tate, through complementation, active replication of endoge-
In this report, we present evidence that infection of mice by
an exogenous virus results in the mobilization of complete
endogenous retroviruses. This includes proviruses that are se-
verely defective and possess large deletions, as well as provi-
ruses that are full length. Furthermore, the transferred se-
quences are transcribed and packaged into virions released
from the newly infected cells.
MATERIALS AND METHODS
Cells and viruses. NIH 3T3 cells were used for the propagation of viruses and
assays of MuLVs. Mus dunni (21) and Fischer rat embryo (FRE) cells and mink
lung fibroblasts (ATTC CCL64), all of which are devoid of endogenous poly-
tropic proviruses, were used as targets to assess the transfer of endogenous
retroviruses. All cells were maintained in tissue culture media supplemented with
glutamine (2 mM), penicillin (100 units/ml), streptomycin (25 ?g/ml), gentamicin
(50 ?g/ml), and amphotericin B (2 ?g/ml). NIH 3T3 cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated
bovine calf serum. M. dunni and FRE cells were maintained in RPMI supple-
mented with 10% heat-inactivated fetal calf serum, and mink cells were main-
tained in DMEM supplemented with 10% heat-inactivated fetal calf serum. The
ecotropic MuLV F-MuLV 57 (30) was obtained as a virus stock after transfection
of 3T3 cells with a plasmid carrying the provirus.
Mice. The mice utilized in this study were of the NFS/N strain maintained as
an inbred colony at Rocky Mountain Laboratories. The mice were infected by
intraperitoneal injection within 24 h of birth and sacrificed at various times after
inoculation. Spleens and thymuses were removed and dissociated by mincing the
tissue, suspending the minced tissue in medium (DMEM containing 10% bovine
calf serum), and passing it through successively smaller syringe needles (from 18
to 23 gauge). Cells from the tissues were then cocultivated for 12 h with target
cell lines seeded at 105cells per 60-mm tissue culture dish. The target cells were
carried for several (10 to 30) passages before the isolation of cellular genomic
DNA and harvesting of released viruses from the tissue culture media. All animal
procedures were done in accordance with the guidelines of the National Insti-
tutes of Health and Rocky Mountain Laboratories animal care and use commit-
Generation of a genomic DNA lambda library of infected FRE cells and
detection of lambda clones containing polytropic MuLV LTRs. DNA was iso-
lated from a clonal cell line derived from FRE cells coinfected with splenocytes
from an F-MuLV-infected NFS/N mouse 34 days after infection. Approximately
1.5 ? 107cells were suspended in 3 ml of lysing buffer from a DNA isolation kit
(Gentra Systems, Inc.; D5000A), and the DNA was thereafter purified according
to the manufacturer’s instructions. The purified DNA was partially digested with
MboI and sedimented on a 10 to 30% linear glycerol gradient in 100 mM NaCl,
10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The gradient was fractionated, and
a sample of each fraction was analyzed on a 0.4% agarose gel (Seakem GTG).
Fractions containing fragments of approximately 15 to 25 kb were pooled; pre-
cipitated with ethanol; resuspended in 10 mM Tris, 1 mM EDTA; and cloned
into the Lambda Fix vector (Stratagene) according to the manufacturer’s instruc-
tions. The library was screened using a probe to the endogenous polytropic
MuLV LTR that was generated and labeled by chemiluminescence using a PCR
DIG Probe Synthesis Kit (Roche; catalog no. 11 636 090 910). The PCR was
accomplished using a primer set consisting of the forward primer BL8313 (AA
GCTAGCTGCAGTAACGCCATTTTGC) and the reverse primer BL8564RC
(GAGGTGCACAGTGCTCTGG). BL8313 corresponds to bases 2024 to 2050
of the prototypic endogenous PT (polytropic structural subclass) provirus MX33
and primes near the 5? end of the U3 region of the LTR (36). BL8564RC
corresponds to the reverse complement sequence of bases 2301 to 2319 of MX33
and primes within the unique 190-bp insert of the U3 region of the LTR (18).
The template was a plasmid containing an endogenous intermediate polytropic
(iPT) provirus designated NB1, isolated from a lambda library of the NFS/N
mouse genome (9). Development of the filters to identify plaques homologous to
the probe was done according to the manufacturer’s instructions. Lambda DNA
preparations were performed as previously detailed (9).
PCR analyses and DNA sequencing. PCR amplification of LTR U3 sequences
of endogenous polytropic proviruses or virion RNA was accomplished using the
primer set described above (BL8313 and BL8564RC) with the puReTaq Ready-
To-Go PCR system (Amersham Pharmacia Biotech Inc.; no. 27-9558-01) or Pfu
Turbo DNA polymerase (Stratagene; no. 600153) according to the manufactur-
er’s instructions. RT-PCR amplification was accomplished using SuperScript III
reverse transcriptase (Invitrogen; no.18080-044), followed by PCR as described
above. The generation of RT-PCR products was dependent upon reverse tran-
scriptase and was unaffected by treatment with DNase, indicating that the tem-
plate corresponded to virion RNA rather than a DNA contaminant. PCR am-
plification and nucleotide sequencing of proviruses in genomic DNA was
accomplished using a set of 39 reverse primers and 40 forward primers approx-
imately evenly distributed across a consensus polytropic provirus genome. This
consensus sequence was constructed from alignments of polytropic proviruses
detected in the Mus musculus genome (38). Similarly, lambda DNA containing
polytropic MuLV sequences was sequenced using this battery of primers.
Nucleotide sequence accession numbers. The sequences of the mobilized
polytropic viruses characterized from FRE clonal cell lines 5, 15, and 51 (see Fig.
3 and 4) have been deposited in GenBank under accession no. FJ544576,
FJ544577, and FJ544578, respectively.
Detection of NFS/N endogenous polytropic LTR sequences
in the genomic DNA of rat cell lines cocultivated with F-
MuLV-infected mouse tissues and in virion RNA released
from the cells. Nearly all polytropic proviruses of NFS/N mice
contain a unique insert of 190 to 212 bp in the U3 region of
their LTRs that is not found in any of the exogenous MuLVs
(18). In this regard, although recombinant polytropic MuLVs
that contain sequences derived from endogenous polytropic
gag, pol, and env genes have been described (8), no recombi-
nant MuLVs have been identified that contain an endogenous
polytropic LTR. We utilized this feature to assess the transfer
of mouse endogenous LTR sequences to target cell lines. The
transfer of such sequences might signal the transfer of intact
polytropic proviruses. Newborn mice were infected with F-
MuLV, and after approximately 7 weeks, the splenocytes or
thymocytes of the infected mice were cocultivated overnight
with FRE cells, which are devoid of endogenous polytropic
proviruses. After cocultivation, the FRE cells were transferred
through multiple (10 to 30) passes to eliminate residual tissue
cells from the mouse. Cellular genomic DNA was then pre-
pared from the cocultivated cells and assayed for the presence
of the endogenous NFS/N polytropic U3 region. This was ac-
complished by utilizing a PCR with a forward primer (BL8313)
near the beginning of the U3 region and a reverse primer
(BL8564RC) within the unique insert of endogenous poly-
tropic proviral LTRs.
Three structural subclasses of polytropic proviruses termed
the PT, the modified polytropic (mPT), and the iPT proviruses
have been identified in NFS/N mice (9, 36). Each subclass is
distinguishable by characteristic features of the U3 region of its
LTR. As a result, three sizes of amplicons from the PCR
analyses might be expected, depending on the structural sub-
class of the polytropic provirus (Fig. 1). No PCR products were
detected using genomic DNA of FRE cells or of FRE cells that
2430EVANS ET AL.J. VIROL.
had been cocultivated with splenocytes from an uninfected
NFS/N mouse (Fig. 1A). In contrast, PCR products were
readily detected in FRE cells that had been cocultivated with
splenocytes from NFS/N mice infected with F-MuLV (Fig. 1B).
The products included one major product of approximately
250 bp and two less prominent bands corresponding to prod-
ucts of approximately 300 and 350 bp. Sequence analysis of the
prominent 250-bp product revealed that it corresponded to the
expected product of the LTR of the PT subclass of endogenous
NFS/N proviruses, while analyses of the minor bands were
inconclusive. Very similar results indicating the transfer of
polytropic LTRs from infected mouse splenocytes were ob-
served from several mice of the same age or older using M.
dunni or mink lung fibroblasts as target cells; both of which are
devoid of endogenous polytropic proviral sequences. The
transfer of LTR sequences was not observed in cocultivation
experiments using thymocytes from F-MuLV-infected NFS/N
mice (data not shown). The spleen, rather than the thymus, is
the major target tissue for F-MuLV.
It was of interest to determine if the newly acquired provi-
ruses in the FRE cells were transcribed and packaged into
virions released from those cells. Virions released from cells
were harvested, and their RNAs were analyzed by RT-PCR for
the presence of NFS/N polytropic LTR sequences. The RNA
from virions released from the cocultivated cells did indeed
contain LTR U3 sequences from NFS mice (Fig. 1C), which
was confirmed by sequence analyses.
Analyses of genomic DNA from clonal lines of FRE cells
cocultivated with F-MuLV-infected NFS/N splenocytes. Nu-
merous colonies of the FRE cells were isolated, and their
DNAs were analyzed for the presence of NFS/N polytropic
LTRs to determine the frequency of infected target cells.
Analyses of 116 clones from FRE cells cocultivated with
splenocytes from two different NFS/N mice identified eight
target FRE single-cell colonies containing an endogenous
LTR from the infected NFS/N mouse. Each clonal line pos-
itive for an NFS/N-derived LTR contained only a single type
of polytropic LTR (Fig. 2). The transferred LTRs detected
in the clonal cell lines corresponded to LTRs from the PT
and mPT subclasses of polytropic proviruses, as was evident
from the sizes of the amplicons (Fig. 1A; see Fig. 4), as well
as by sequence analyses (data not shown). Six clonal cell
lines contained PT LTRs, and two contained mPT LTRs.
LTRs from the iPT class of proviruses, which comprise a
small proportion of NFS/N endogenous proviruses (Fig. 1),
were not detected. The observation that each clonal line
contained a single type of polytropic LTR (Fig. 2), coupled
with the finding that about 7% of the clonal lines tested
FIG. 1. PCR amplification of the LTR U3 region of endogenous
polytropic proviruses detected in FRE cells cocultivated with spleno-
cytes from F-MuLV-infected NFS/N mice and RT-PCR amplification
of virion RNA released from the cocultivated FRE cells. The sche-
matics depict the U3 regions of the LTRs of the respective endogenous
polytropic provirus subclasses. Adjacent blocks indicate direct repeats,
whereas isolated blocks indicate regions that are repeated in the U3
regions of other retroviruses. Deletions relative to the U3 regions of
other viruses are depicted by the breaks in the lines connected by the
downward extended Vs. The inserts ranging from 190 bp to 212 bp are
indicated by the thick horizontal lines. The annealing sites of the
forward and reverse primers are indicated by arrows, and the amplicon
sizes are indicated to the right of the schematics. (A) Lane 1, 100-bp
ladder; lane 2, PCR products from genomic DNA of uninfected FRE
cells; lane 3, PCR products from genomic DNA of FRE cells coculti-
vated with splenocytes from an uninfected NFS/N mouse (77 days old).
(B) Lane 1, 100-bp ladder; lane 2, PCR products from genomic DNA
from FRE cells cocultivated with splenocytes from an NFS/N mouse
infected with F-MuLV (34 days old). (C) Lanes 1 and 2, RT-PCR
products from virion RNA released from FRE cells cocultivated with
splenocytes from two different NFS/N mice infected with F-MuLV (34
days old); lane 3, 100-bp ladder.
FIG. 2. PCR amplification of the LTR U3 region of endogenous
polytropic proviruses from clonal cell lines of FRE cells derived from
cells cocultivated with F-MuLV-infected NFS/N splenocytes. Lane 2,
100-bp ladder; lanes 1 and 3 to 8, PCR products from the DNAs of
seven clonal cell lines. The cell lines in lanes 3 and 4 tested positive for
an mPT LTR and a PT LTR, respectively. The cell lines in lanes 5 to
7 tested negative for polytropic LTRs, and the cell line in lane 7 tested
positive for a PT LTR.
VOL. 83, 2009 MOBILIZATION OF ENDOGENOUS RETROVIRUSES2431
positive for a polytropic LTR, suggested that most of the
positive clones harbored a single polytropic provirus.
LTRs detected in FRE cells cocultivated with F-MuLV-in-
fected NFS/N splenocytes signal the transfer of intact endog-
enous mouse proviruses. The detection of polytropic LTRs in
the DNA of cocultivated FRE cells strongly suggested that
endogenous proviruses from the mouse had been transferred
to the rat cells. Alternatively, the detected proviruses could
correspond to a new type of recombinant between the exoge-
nous ecotropic retrovirus and the endogenous polytropic pro-
viruses that retained the polytropic LTR. To distinguish be-
tween these possibilities, a lambda library was generated from
the genomic DNA of one of the clonal cell lines that harbored
a provirus containing a polytropic LTR, and the library was
subsequently screened for lambda clones containing polytropic
LTR sequences. Three lambda clones containing the poly-
tropic LTR were isolated from the library and subjected to
sequence analysis. One of the clones contained an entire pro-
virus, while the provirus sequences of the other two clones had
been truncated during the cloning process and retained only
the 3? region of the proviral genome. Sequence comparisons of
the proviruses and their flanking sequences indicated that the
three clones corresponded to clones of the same integrated
The provirus transferred to the rat cells did not contain
discernible ecotropic F-MuLV sequences, indicating that it
corresponded to a complete endogenous NFS/N provirus
rather than a new type of recombinant virus. It exhibited sev-
eral deletions that would render the virus replication defective,
including a large deletion encompassing nearly all of the env
gene sequences (Fig. 3).
The 3?-flanking sequences of all three lambda clones were
identical and were located at the same position on chromo-
some 11 of the rat genome. This corroborated our conclusion
from the sequence data that the clones corresponded to the
same integrated provirus that was precisely integrated into the
rat genome rather than being the result of the fusion of mouse
and rat cells or of inadvertent transfection of the rat cells by
mouse DNA. Furthermore, the provirus exhibited the con-
served 3? CA dinucleotide proviral terminus immediately ad-
jacent to rat sequences, typical of integrants mediated by
MuLV (Fig. 3).
PCR analyses of the genomic DNA from several additional
FRE clonal cell lines that harbor a single polytropic LTR
identified two proviruses closely corresponding to previously
characterized NFS/N proviruses. These proviruses corre-
sponded to NE1 and NP1, both of which are PT proviruses
(Fig. 4). The transferred proviruses differed by only one or two
env gene nucleotides, respectively, from the previously charac-
terized NFS/N proviruses (9). These differences likely reflect
FIG. 3. Structure of an NFS/N polytropic provirus transferred to FRE cells and its integration junction with rat genomic sequences. A provirus
was isolated from a lambda library derived from a clonal cell line of FRE cells (clone 5) that had been cocultivated with splenocytes from an NFS/N
mouse 34 days after infection with F-MuLV. The structure was derived from an alignment of the nucleotide sequence of the transferred provirus
with an endogenous polytropic provirus consensus sequence. An apparent insertion of 17 A residues occurred at the position of a small deletion
from 2494 to 2557. Large deletions are indicated by dashed lines extending down from the bar diagram. The proviral DNA 3? terminus and flanking
sequence present in a FRE clonal cell are indicated in the expanded region. FRE rat sequences are shaded. Identical sequences were obtained
from three individual lambda clones from a genomic library of the clonal cell line.
2432 EVANS ET AL.J. VIROL.
point mutations incurred during retroviral replication subse-
quent to mobilization by F-MuLV. Both of these proviruses
contain intact env genes, and one, NP1, is a frequent partici-
pant in recombination with ecotropic MuLVs (1).
The transfer of intact endogenous mouse proviruses occurs
by pseudotyping within ecotropic MuLV virions. Mice infected
with F-MuLV express both ecotropic and polytropic Env pro-
teins. The transfer of the endogenous polytropic retroviruses
of mice to rat cells could occur through infection utilizing
polytropic Env proteins or, alternatively, through pseudotyping
of polytropic genomes within ecotropic F-MuLV virions (10,
24, 34). Pseudotyping of the NFS/N endogenous retrovirus
genomes was assessed by comparing the transfer of proviruses
to uninfected FRE cells to the transfer of proviruses to FRE
cells chronically infected with F-MuLV. Viral interference ren-
ders F-MuLV-infected cells refractory to reinfection by eco-
tropic virions, including endogenous proviruses pseudotyped
by F-MuLV. Thus, pseudotyping by the ecotropic virus would
be reflected in a decrease in the transfer of endogenous ge-
nomes to F-MuLV-infected FRE cells compared to uninfected
FRE cells. For these experiments, we utilized young mice, 10
days postinfection, to minimize the level of replication-compe-
tent recombinant polytropic MuLVs that also might be capable
of pseudotyping endogenous retrovirus genomes. PCRs re-
vealed that the proviruses were readily transferred to unin-
fected FRE cells. In contrast, the F-MuLV-infected cells were
nearly impervious to the transfer of mobilized endogenous
polytropic genomes, indicating that the polytropic genomes
released from the mouse splenocytes were extensively
pseudotyped (Fig. 5).
Recombination between exogenous ecotropic MuLVs and
endogenous polytropic MuLVs, a process that requires full-
length transcription of the participating endogenous polytropic
provirus, occurs quite frequently in infected mice. Recombina-
tion occurs during RT of the heterodimeric RNA; thus, the
FIG. 4. Structures of two NFS/N polytropic proviruses transferred to FRE cells. The proviruses were sequenced from PCRs of genomic DNAs
from clonal FRE cell lines each containing a single NFS/N polytropic provirus. The structures were derived from alignments of the nucleotide
sequences of the transferred proviruses with an endogenous polytropic provirus consensus sequence. It is nearly certain that the LTR sequences
were derived from both the 3? and 5? LTRs, although the presence of both complete LTRs could not be formally determined by these analyses.
With that qualification, we have depicted the proviruses as complete structures possessing both LTRs. The provirus found in clone 15 contains a
deletion of 100 bp in the Gag region. It differs from the NP1 proviral env gene found in the NFS/N genome by only 1 base at position 7184 in the
consensus sequence. The provirus found in clone 51 contains three small deletions in the Gag region and differs from the NE1 proviral env gene
found in the NFS/N genome by a single mismatch at residue 6394 in the consensus sequence.
VOL. 83, 2009MOBILIZATION OF ENDOGENOUS RETROVIRUSES 2433
full-length copy of the endogenous provirus is capable of being
packaged within virions. We surmised that if full-length tran-
scripts of the endogenous polytropic proviruses are packaged
as RNA heterodimers, it is likely that they are also packaged as
RNA homodimers that might subsequently be transferred to
different cells in the absence of recombination. The transfer of
the endogenous proviruses was signaled in our analyses by
detection of the endogenous polytropic U3 regions in target
FRE cells. This region has never been detected in a recombi-
nant polytropic virus and would likely be indicative of transfer
of the entire endogenous genome. This was confirmed by anal-
ysis of the complete genomes of transferred proviruses. Fur-
thermore, analyses of flanking genomic sequences in the target
rat cells suggested that the transferred NFS/N provirus had
inserted into the rat genome in a manner typical of MuLV-
We have recently characterized many of the endogenous
retroviruses of NFS/N mice and noted some features of these
proviruses that suggest that the proviruses had undergone ac-
tive replication during recent periods of their evolution (9). In
this regard, one of the endogenous polytropic MuLVs was
clearly a recombinant between two distinct classes of the en-
dogenous viruses. Since recombination occurs through the co-
packaging of the RNAs of each parent in a heterodimeric
RNA, this observation strongly suggested that both types of
viruses had simultaneously replicated in the same cell, recom-
bined, and subsequently reintegrated into the germ line. A
second observation suggesting active replication of the endog-
enous viruses was the presence of identical lethal mutations in
different proviruses. This suggested that a replication-defective
endogenous retroviruses was transcribed and pseudotyped
within a replication-competent retrovirus and subsequently re-
retroviruses may have been initiated through widespread infec-
tion of mouse populations by exogenous retroviruses capable of
circumventing restriction factors present in the mouse.
Recombinant viruses released from mice infected with the
ecotropic MuLVs are frequently pseudotyped within ecotropic
virions (10, 24, 34). It seems likely that this might also be the
case with the transfer of the endogenous proviruses, and cer-
tainly with those proviruses that exhibit deletions rendering
them replication defective. Indeed, our observation that the
transfer was blocked in target cells chronically infected with
the ecotropic F-MuLV confirmed that the transfer of endog-
enous proviruses to target cells was facilitated by viral
pseudotyping. Pseudotyping may provide an effective means to
evade host replication restriction factors that have been cir-
cumvented by the ecotropic virus. Our studies suggest that the
packaging and infectious transfer of endogenous retroviruses
after infection of the host by exogenous retroviruses may be a
We have demonstrated that the proviruses transferred to the
FRE target cells were transcribed and released into progeny
virions, indicating the potential for further spread. Further-
more, the transfer of endogenous viruses after infection by
exogenous viruses occurs early after infection, before recom-
binant viruses are detected, and may represent the major in-
teraction of exogenous viruses with endogenous retroviruses.
The finding that endogenous viruses are mobilized by infection
of the host with exogenous viruses may have far-reaching im-
plications in retrovirus evolution, as well as in retroviral patho-
genesis. The spread of intact endogenous proviruses to cells of
other species strongly suggests that a similar spread is occur-
ring in the infected host, particularly early after infection, when
inhibition by viral interference would be minimal. The expres-
sion of viral gene products has been implicated in pathological
processes, such as the involvement of polytropic SU proteins in
autoimmune murine lupus nephritis (2). The spread of endog-
enous proviruses in the host could potentially facilitate the
inappropriate expression of viral proteins, which could in turn
trigger a pathological response. Furthermore, retroviral spread
can result in insertional mutagenesis, as well as transcriptional
activation, in infected cells. Clearly, the mobilization of one
virus by another is clinically relevant and could contribute to
the pathogenicity of exogenous retroviruses, such as the
MuLVs, human immunodeficiency virus, and human T-cell
We thank A. Kolokithas, K. Peterson, and K. Hasenkrug for helpful
This research was supported by the Intramural Research Program of
the NIAID, NIH.
1. Alamgir, A. S., N. Owens, M. Lavignon, F. Malik, and L. H. Evans. 2005.
Precise identification of endogenous proviruses of NFS/N mice participating
FIG. 5. Block in the transfer of NFS/N polytropic virus to FRE
cells by retroviral interference. FRE cells or FRE cells chronically
infected with F-MuLV were cocultivated with splenocytes from NFS/N
mice inoculated with F-MuLV or from uninfected NFS/N mice as
controls. The U3 region of the LTR was amplified by PCR to assess the
transfer of NFS proviruses to the FRE cells. Lane 1, 100-bp ladder;
lane 2, FRE cells cocultured with 10-day-old F-MuLV-infected NFS
mouse splenocytes; lane 3, F-MuLV-infected FRE cells cocultured
with 10-day-old F-MuLV-infected NFS mouse splenocytes. PCRs per-
formed with DNA derived from cocultivated FRE cells show robust
amplification of both polytropic (?250-bp) and modified polytropic
(?300-bp) LTRs (lane 2). The PCRs performed with DNA from
cocultivated FRE cells that had been previously infected with F-MuLV
exhibited a striking decrease in the products of both polytropic LTRs
2434 EVANS ET AL.J. VIROL.
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