Rev. sci. tech. Off.
int.
Epiz., 1998,17 (1), 269-277
Fv1,
the mouse retrovirus resistance gene
J.P. Stoye
Division of
Virology,
National Institute for Medical Research, The Ridgeway, Mill
Hill,
London NW7
1
AA,
United Kingdom
Summary
A number
of
genes which affect
the
susceptibility
of
mice
to
infection
by
retroviruses have been described. One of the most interesting of these genes
is
Fvl (Friend virus susceptibility 1), which acts
at a
stage in the retroviral life-cycle
following virus entry into the cell but prior to integration and formation
of
proviral
structures.
A
detailed understanding
of
the mode
of
action
of Fv1
might
be
expected to shed fresh light on early steps of the retroviral replication, although
progress has been slow in this area due to uncertainty about the nature of the
Fv1
gene.
The recent cloning
of Fvl
by
a
positional approach fills this gap
in
current
knowledge.
Fv1 appears to be derived from
a
fragment of
a
retroviral genome,
an
observation that may suggest novel approaches
to the
control
of
retroviral
replication.
Keywords
Dominant negative mutation
-
Endogenous retroviruses
-
Genetics
-
Friend virus
susceptibility-1
-
Integration
-
Positional cloning
-
Provirus
-
Retroviridae.
Discovery of retroviral
resistance genes in mice
Retroviral
infection can have a variety of severe consequences,
which include oncogene activation leading to cancer,
cell
death
leading to immunosuppression and germ-line
integrations leading to inherited mutations.
Since
eukaryotic
evolution has taken place against a background of retroviral
infections,
it would be surprising if genetic mechanisms for
controlling retrovirus replication had not evolved.
Studies
of leukaemogenesis in mice performed in the
1960s
and
1970s
provided ample evidence to
support
this idea.
Many
of these studies utilised the Friend virus (Fv) complex,
an immunosuppressive virus preparation which rapidly
induces malignant erythroleukaemias (38). A number of
genes controlling responses to Friend virus infection were
identified (36), including at least five genes controlling
immunological
responses as well as six genes for Friend virus
susceptibility,
known as
Fvl-Fv6,
which act by
non-immunological means
(20).
Resistance
genes were also identified by in
vitro
studies. For
example,
it was shown that murine leukaemia viruses
(MLV)
could
be sub-divided into three categories based on their
ability
to grow on
cells
derived from the embryos of different
strains of mice
(Table
1). Some viruses, termed N-tropic,
yielded higher titres on
cells
from the National Institutes of
Health (NIH)-Swiss strain (N)
than
on
cells
from
BALB/c
(B)
mice,
while B-tropic viruses grew better on
B-
than
on N-type
cells
(19).
A third category of virus, NB-tropic, grew equally
well
on N- and B-type
cells.
Both
N- and
B-tropic
viruses grew
poorly on
cells
from Fl hybrids, which implies that resistance
was dominant to susceptibility. Subsequent genetic studies
showed that the in
vitro
restriction was controlled by a single
genetic
locus (32), which proved to be identical to the Fvl
locus
identified in vivo
(31)
and
mapped
to the distal region of
mouse chromosome 4
(35).
Table
I
The effect of
FV1
in vitro
Virus n/n Genotype of cells
n/b b/b
N-MLV
100 1 1
B-MLV
1 1 100
NB-MLV 100 100 100
a) Relative titres
in
plaque assay (absolute titres
are
usually approximately
105 and
103
infectious units/ml in permissive and restrictive cells, respectively)
N
:
National Institutes of Health-Swiss strain
B
:
BALB/c strain
MtV
:
murine leukaemia virus
Although the block to infection mediated by Fvl is not
absolute,
in
vitro
titres are reduced by a factor of one
hundred
to one thousand (19), and Fvl can confer resistance to
270 Rev. sci. tech. Off.
int.
Epiz.,
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(1)
naturally-occurring MLV-induced disease
(18).
The majority
of
inbred strains of
mice
carry either the n or the b allele of Fvl
(25),
though
two other restrictive
alleles,
with subtly different
phenotypes, have also been described (4,
28).
Most wild mice
do not appear to exhibit Fvl restriction
(28).
To date, there is
no convincing evidence for the presence of Fvl restriction in
other species of animal.
Phenotypic characterisation of
Fv1 restriction
The
observation that Fvl restriction is manifested in
vitro,
in a
dominant fashion, implies that this restriction has a direct
effect
on retroviral replication, most likely mediated by an
interaction between the Fvl gene
product
and some
component of the virus. Initial mechanistic studies were
therefore focused on discovering the stage in the retroviral
life-cycle
at which it might be acting and in determining the
viral target for restriction.
The
retroviral
life-cycle
is shown in schematic form in
Figure
1. Readers are referred to Coffin (10) for a review of
retroviral replication. Retroviruses are enveloped viruses
containing single-stranded RNA as their genetic material.
Extracellular
virus binds to a
specific
receptor on the target
cell
by means of the envelope protein. Following membrane
fusion, the viral capsid enters the
cell.
Reverse transcription
of
the viral RNA takes place to form linear double-stranded
DNA.
This DNA, still associated with remnants of the capsid,
moves to the nucleus where integration of viral DNA into the
host
cell
DNA occurs, which results in the formation of a
provirus. This DNA is transcribed and the resulting RNA is
translated by the synthetic machinery of the
cell.
Viral RNA
and protein assemble and bud
through
the
cell
membrane,
thereby acquiring an envelope. Proteolytic cleavage then
results in virion maturation.
Initial
studies showed clearly that Fvl did not
affect
the entry
of
virions into
cells.
Mixed viral pseudotypes can be formed
between rhabdoviruses, such as vesicular stomatitis virus, and
retroviruses (both of which are enveloped RNA viruses).
Preparations of vesicular stomatitis virus genomes coated with
the envelope protein from an N-tropic
MLV
grew equally well
on N- or B-type
cells
(22, 30).
Since
replication of vesicular
stomatitis virus was
dependent
on the activity of the MLV
envelope,
this experiment shows that Fvl must act at a stage
in the retroviral
life-cycle
subsequent to the stages mediated
Fig.
1
Possible sites of action for the Fv1 gene product
The retroviral life-cycle is shown in schematic form illustrating the points at which the
Fv1
gene product might act
Rev. sci. tech. Off.
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17 (1) 271
by
the envelope protein, in other words at either attachment
to the cellular receptor or uptake into the
cell.
A
variety of experimental approaches have ruled out the
possibility
of Fvl action late in the viral replication
cycle.
Immunofluorescence
studies showed greatly reduced levels of
viral protein in infected
cells,
implying that restriction must
act
at, or before, translation of novel viral products (19).
Nucleic
hybridisation experiments showed reduced levels of
new viral transcript in restricted
cells,
which points to a
preintegration or transcriptional block (27). Finally, direct
measurements of the amount of integrated viral DNA also
showed reduced levels, thereby indicating that Fvl must act
prior to integration and formation of proviruses
(26).
Attempts to define more precisely which of the preintegration
steps (reverse transcription, nuclear import and integration) is
affected
by Fvl action have yielded conflicting results. Early
experiments showed no reduction in the amounts of freshly
synthesised viral DNA in the cytoplasm of infected
cells
(26),
but this appears to depend on both the time at which
measurements are made and the
cell
type examined (33).
Nuclear association of only slightly reduced levels of viral
DNA
has been reported, but it is not clear whether that DNA
was truly within the nucleus
(33).
Preintegration complexes
isolated
from restricted
cells
appear to have normal
integration activity in
vitro,
which suggests that no overt
damage to the complex results from the interaction with Fvl
gene product (33). Thus, only careful measurements of the
amounts and precise localisation of the preintegration
complex
in restricted
cells
seem likely to resolve these issues.
Attempts to answer the question regarding the target for Fvl
restriction have been more successful. Initial studies showed
that changes in the capsid protein (also known as p30 or CA),
the major protein component of the viral capsid, were
associated
with changes in viral tropism
(21,34).
Studies with
'mix
and match' recombinants made using cloned viral DNAs
confirmed
these observations and showed that the viral
determinants of Fvl could be mapped to a pair of amino acids
in CA (Fig. 2) (8, 13). Very recent studies performed using
site-directed mutagenesis reveal that the second of the two
amino acids (arginine/glutamate) is the more important in
determining tropism
(29).
The
fact
that CA is the target for the Fvl gene product is fully
consistent
with current understanding of the site of action of
Fvl,
since CA is known to be present on the subviral complex
in which reverse transcription occurs and which mediates
nuclear import and integration
(9).
However, further progress
in understanding how Fvl might act was inhibited by a
complete
absence of information about the Fvl gene product
and for this reason, the keen interest that was shown in-Fvl in
the late
1970s
and early
1980s
has waned in recent years.
Cloning of Fvl
How might the cloning of Fvl be approached? One method
would be to try to take advantage of what little is known of
Fvl
functional properties and to isolate a cellular protein
which binds to
MLV-CA,
or to clone a gene encoding such a
protein. Several laboratories have attempted to clone Fvl
using the so-called yeast two-hybrid system (14), which
N
:
National Institutes of Health-Swiss strain
B
:
BALB/c strain
Fig.
2
Viral determinants of Fv1 tropism
Simple retroviruses such as murine leukaemia virus (MLV) possess three genes. These are called gag, which encodes the components of the viral capsid
MA (matrix), p12, CA (capsid), NC (nucleocapsid); pol, which provides PR (protease), RT (reverse transcriptase) and IN (integrase); and env, which encodes the
envelope proteins SU (surface) and TM (transmembrane). The primary target for the
Fv1
gene product is amino acid 110 of CA-arginine in
N-MLV
and glutamate
In
B-MLV
272 Rev. sci. tech. Off.
int.
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17 (1)
allows
the detection of interacting proteins expressed from
complementary DNAs
(cDNAs)
in yeast. These attempts have
not met with success,
perhaps
because the CA 'bait' used in
these experiments did not assume the correct conformation
for
interaction with the Fvl gene
product.
An alternative approach, which ultimately proved successful,
was to
adopt
a genetic approach known as positional cloning.
In
essence, positional cloning comprises three steps. Firstly,
the gene in question is mapped as accurately as possible to a
small,
defined genetic interval. Then, the DNA from this
interval is cloned. Finally, the genes encoded within these
clones
are identified and differences between different alleles
are sought. Though labour-intensive, this approach has met
with considerable success in cloning genes associated with
inherited diseases in man and in a variety
of
mouse mutations,
in which little was known about the function of the mutated
gene
(11).
The
possibility of cloning Fvl in this way arose as a result of
observations made in the course of genetic studies with the
endogenous
MLVs
of
mice.
If a retrovirus infects a germ
cell,
the resulting provirus can
Become
part
of the germ line,
provided that
undue
harm
to the host is not caused by virtue
of
expression or by the position of the provirus in the genome.
These
inherited elements, which are known as endogenous
retroviruses, are associated with a plethora of biological
phenomena (7). The endogenous retroviruses of a given
species
can be classified into a few groups, each containing up
to ten thousand individual members which show a high
degree of nucleotide sequence similarity but differ in their
specific
integration sites within the genome. A lower degree of
sequence
similarity is seen between different groups. There
are between
fifty
and one
hundred
endogenous
MLVs
in mice
(7).
In the course of
mapping
one MLV sub-family, four
proviruses, called Xmv8, Xmv9,
Xmvl4
and
Xmv44,
were
shown to be tightly linked to Fvl (15).
Since
the Fv4
restriction gene was known to correspond to a fragment of a
retrovirus, which prevented infection by competing for the
receptor for virus binding
(24),
it was suggested that one or
more of these endogenous proviruses might correspond to
Fvl
(15). As described below, this suggestion,
though
not
correct
in detail, contained an element of
truth.
To
test the possibilities that one of these proviruses might be
Fvl
(or
if
not Fvl itself, might lie
close
enough to Fvl to allow
positional cloning of the
gene),
a detailed genetic analysis of
the region of distal chromosome 4 known to contain Fvl was
performed
(37).
This
study
ruled
outXmv8,
Xmv9 and
Xmv44
as candidates for Fvl. However, Xmv9 and two other markers
(Nppa
and
lap3rcl
1) showed no recombination with Fvl, and
the researchers argued that these markers must lie within
1.2 megabase (Mb) of one another and Fvl, confirming the
feasibility
of a positional approach.
The
research team set out to clone the chromosomal interval
spanning these markers. Genomic libraries containing large
fragments of DNA derived from
C57BL
mice (Fvl
b/b)
cloned
as yeast artificial chromosomes
(YACs)
were screened for
Nppa
and Xmv9. A variety of different
YACs
were isolated.
Unfortunately, attempts to generate an ordered array of
overlapping
YACs
failed because many of the
YACs
were
unstable, probably as a result of multiple copies of a repeated
zinc
finger gene.
At
this point, the research team decided to abandon a
systematic
gene-finding approach and. concentrate on testing
the cloned
YACs
for Fvl activity in a functional test (Fig. 3),
based on the argument that since Fvl was a dominant gene,
introduction of a
YAC
containing Fvl into
cells
should result
in an alteration of their susceptibility to
MLV
infection. Mouse
cells
were fused with yeast carrying Xmv9 or
Nppa
YACs
and
stable
clones were isolated. These
cell
clones were expanded
and infected with N- and
B-tropic
viral vectors carrying a gene
for
resistance to the antibiotic puromycin. By enumerating
puromycin-resistant colonies, the
cells
could be typed for Fvl
phenotype. Using this approach, a YAC carrying the
Nppa
gene was shown also to contain Fvl activity
(5).
As expected,
given the
Fvlb/b
origin of the
YAC,
the introduced resistance
gene affected N-tropic virus replication but did not inhibit an
NB-tropic
virus.
To
isolate the Fvl gene, a cosmid library was
prepared
from
the
YAC
and individual cosmids carrying Fvl were identified
in the functional assay. Fragments of a positive cosmid were
retested and a 6.5 kilobase (kb) Fvl-positive clone was
isolated.
The DNA sequence of this clone was determined and
a
1.4 kb open reading frame with a predicted protein
product
of
459 amino acids, subsequently shown to possess Fvl
activity
restricting the replication of B-tropic virus, was
identified. The corresponding fragment was cloned from a
library
prepared
from an Fvln/n mouse restricted B-tropic
MLV,
thus
confirming that the research team had succeeded
in cloning Fvl. The predicted
product
of the n allele is
nineteen amino acids shorter
than
that of the b
allele;
the
alleles
also differ at two other amino acid positions (5).
Origin
of
Fv1
Analysis
of the structure and distribution of Fvl revealed a
number of
unusual
features (Fig. 4) (5, Le
Tissier
et al, in
preparation). Firstly, most genes have multiple exons,
whereas Fvl comprises a single long exon. Secondly, the
presumptive polyadenylation signal is provided by the second
of
a pair of short, interspersed nuclear repeats (B2 elements)
located
downstream of the open reading frame, rather
than
by
a
'natural' gene-specific signal. Thirdly, the Fvl gene is present
only
in mice and not in rats or humans, which implies a
relatively
recent evolutionary origin.
Sequence
data
bank comparisons provide an explanation for
these findings. The Fvl coding sequence is related to the gag
gene of the endogenous retrovirus family, called
HERV-Ls
or
Rev. sci. tech. Off.
int.
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17 (1) 273
Normal cells (Fv1n) Mouse cells fused with YAC carrying
Fvf
Fvl
:
Friend virus susceptibility
1
MLV
:
murine leukaemia virus
N
:
National Institutes of Health-Swiss strain
B
:
BALB/c strain
YAC
:
yeast artificial chromosome
Puro
:
puromycin
Fig.
3
Functional cloning of Fvl
The assay used for detecting the Fvl gene in cloned DNA is illustrated in schematic
form.
YACs carrying the NeoR gene were introduced into mouse cells by
spheroplast fusion (23), and G418 resistant cell clones were isolated. These cells were then infected with equal titres of
N-tropic
or
B-tropic
retroviral vectors
carrying the
PuroR
gene. Reduction in the number of puromycin-resistant colonies as a result of fusion with a specific YAC was indicative of the presence of Fvl
on
that YAC
MuERV-Ls,
which are themselves highly divergent from MLV
(3,
12). The
Fvl
gene seems to have resulted from the
insertion of all or
part
of such an element into a
Mus
progenitor. Expression is dependent on the chance presence
on either side of the insertion point of cryptic signals.
Transcription utilises a cellular promoter found upstream of
the open reading frame and transcripts are polyadenylated at
an insertion element.
Significance and future
prospects
What
are the implications of the observation that
Fvl
is
derived from the
gag
gene of an inserted retrovirus? Perhaps
the most interesting feature of this result is that it suggests a
possible
model of action for the gene. Gag proteins must
interact
during
virus assembly, and these proteins have been
shown to bind tightly to one another through complex
interaction domains (1). This suggests that
might be
considered as a dominant negative mutation in which
restriction involves an interaction between the Fvl gene
product and the CA protein present on incoming virions: an
interaction which would result in inhibition of capsid
function
(17).
However, the
fact
that Gag proteins have not
previously been implicated in superinfection resistance
should be noted, which
thus
implies unusual properties for
the
MuERV-L
Gag protein that corresponds to Fvl.
Stoye
and
colleagues
are currently attempting to identify the domains of
the Fvl gene product which interact with MLV in order to
determine whether CA binding can be dissociated from
inhibition of virus replication. If binding alone results in
restriction, the team will attempt to prepare a peptide, or
peptide mimetic, capable of inhibiting virus replication.
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