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Journal of Virology, February 1999, p. 1565-1572, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evidence that the Human Foamy Virus Genome Is
DNA
Shuyuarn F.
Yu,
Mark D.
Sullivan, and
Maxine L.
Linial*
Division of Basic Sciences, Fred Hutchinson
Cancer Research Center, Seattle, Washington 98109
Received 23 December 1997/Accepted 13 November 1998
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ABSTRACT |
The genomes of the spumaviruses, of which human foamy virus (HFV)
is the prototype, are very similar to those of other complex retroviruses. However, in some aspects of the viral replicative cycle,
HFV more closely resembles pararetroviruses such as hepatitis B virus.
Previous work indicated that HFV extracellular particles contain
apparently full-length double-stranded DNA, as well as RNA. We have
further characterized the amount of DNA in particles and the role that
this DNA has in viral replication. Experiments with the reverse
transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) suggest that
reverse transcription is largely complete before extracellular virus
infects new cells. In addition, we have been able to show that DNA
extracted from virions can lead to production of virus after
transfection. Taken together, these data suggest that complete, or
nearly complete, proviral-length DNA is present in viral particles and
that this DNA is sufficient for new rounds of viral replication.
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INTRODUCTION |
Human foamy virus (HFV) is the
prototype virus of the Spumavirus genus of retroviruses. It
was originally designated as a human virus because the first isolation
was from a human cancer-derived cell line (1); however,
recent analyses show that HFV is of chimpanzee origin (11).
The spumaviruses are complex retroviruses whose genomes contain the
three hallmark retroviral genes, gag, pol, and
env, as well as two or more genes encoding nonstructural proteins. One of these is a transactivator protein called Bel1 or Tas,
which is required for transcription from the long terminal repeat (LTR)
and internal promoters and absolutely required for infection (17,
18). The genomic organization of foamy viruses is
identical to that of other retroviruses with respect to the LTRs at the
ends of the proviral DNAs, a primer binding site required for
negative-strand DNA synthesis, and the order of the structural proteins
(reviewed in reference 25). The viral
genomic sequences are so like those of other retroviruses that
there was no reason to suspect that foamy viruses would significantly
diverge from other retroviruses in any aspects of replication.
Therefore, it has been surprising to discover that many details of the
foamy virus replicative pathway substantially differ from those of
conventional retroviruses such as murine leukemia virus or human
immunodeficiency virus (HIV).
In some respects, foamy viruses more closely resemble pararetroviruses
such as hepatitis B virus (HBV). For example, as in HBV, HFV polymerase
protein is not synthesized as a polyprotein containing
structural protein determinants, suggesting that Pol incorporation into
particles occurs in a different manner from that of retroviruses
(6, 16, 34). The Gag protein of HFV is not efficiently
processed by protease (PR) into matrix, capsid, and nucleocapsid (NC)
components (8, 14, 21). Additionally, HFV particles do not
bud from cells unless viral glycoproteins are present (3),
analogous to what is observed for HBV. But perhaps the feature of HFV
that is most divergent from retroviruses is the presence of large
amounts of apparently full-length double-stranded DNA in extracellular
virions, which we found in an earlier study (34). In that
study, we found that long viral DNA was in roughly 10 to 15% of the
viral particles. This is in contrast to the case with human HIV, where
it was shown that while about 0.1 to 1.0% of the virions contain
strong-stop DNA, less than 0.001% of the virions contain full-length
DNA (32) and this DNA is not required for infectivity
(2). This suggested, as in the case of the hepadnaviruses,
that a large amount of reverse transcription is completed before, or
shortly after, the time when virions bud from infected cells. However,
the role of the DNA in HFV replication was not determined in this prior study.
To examine whether the presence of DNA in extracellular virions is
important for viral replication, several experimental approaches were
taken. Using quantitative PCR methods (13), we have been able to show that about 20% of the particles contain long DNA. Some of
the virion-associated DNA is infectious when transfected into tissue
culture cells and can lead to the production of infectious virus.
Finally, the effects seen with the reverse transcriptase (RT) inhibitor
3'-azido-3'-deoxythymidine (AZT) are most consistent with a DNA genome,
in that pretreatment of cells has only a small effect on viral
infectivity but treatment of infected cells abolishes production of
viral infection. Taken together, these data indicate that preformed DNA
is an important component of de novo HFV infection.
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MATERIALS AND METHODS |
Tissue culture methods.
Wild-type HFV with a deletion
of the unique 3' (U3) region of the LTR, called HFV13, is derived from
the molecular clone pHSRV13 (18). pHSRV13 DNA is
transfected into BHK cells. After extensive cytopathic effects are
noted (5 to 7 days after transfection), cell lysates and viral
supernatants are combined and used to infect diploid human embryonic
lung (HEL) fibroblast cells as previously described (36).
For some experiments, virus was harvested from the supernatant of
chronically infected H92 human erythroleukemia cells (37).
Viral infectivity assays using FAB cells (BHK cells containing HFV
LTR-
-Gal DNA) were done as previously described (36)
except that cells were stained with X-Gal
(5-bromo-4-chloro-3-indolyl--D-thiogalactopyranoside) for 4 h at 37°C and then left at room temperature overnight
before blue cells were counted. FAB and BHK cells were grown in
Dulbecco modified Eagle (DME) medium with 5% fetal calf serum, HEL and NIH 3T3 cells were grown in DME medium with 10% fetal calf serum, and
H92 cells were grown in RPMI medium with 5% fetal calf serum. Transfection and virus harvest were done as described for HFV13. A
Moloney murine leukemia virus (MLV) vector, LNCZ, containing the
-galactosidase (-Gal) gene (19), was packaged with MLV Gag and Pol proteins and the vesicular stomatitis virus (VSV) G protein
(33) and obtained from Dusty Miller (Fred Hutchinson Cancer
Research Center). MLV(VSV) infections were done in the presence of
8 µg of Polybrene per ml.
For treatment with inhibitors, FAB cells were pretreated for 4 h
with inhibitor and then infected with cell-free virus stocks suspended
in medium containing inhibitors. Virus was left on the cells for 4 or
24 h, and then the medium was changed to fresh medium containing
inhibitor. The cells were fixed at different times after infection and
stained for -Gal activity. Individual H92 cell clones derived from a
culture persistently infected with HFV (37) were
continuously cultured with 100 µM AZT for several months.
Periodically, cell lysates were made and combined with culture
supernatants and assayed on FAB cells for infectious virus. AZT was
from Sigma, ddI was from L. Corey, (Fred Hutchinson Cancer Research
Center), and lamivudine (3TC) was from R. Schinazi (Emory University,
Atlanta, Ga.).
Transfections were done with Lipofectamine or Lipofectamine Plus (GIBCO
BRL, Gaithersburg, Md.). FAB cells (1.2 × 10
5) were
plated in 35-mm-diameter tissue culture dishes. The next
day, the cells
were transfected in accordance with the manufacturer's
directions. The
transfection mixture contained 5 µl of Lipofectamine
and different
amounts of virion DNA extracted from the cell-free
supernatants of
acutely infected HEL cells or chronically infected
H92 cells. DNA was
left on the cells for 3 h. A linear fragment
of pHFV13 digested
with
EagI and
SalI (12,070 bp) was used as
a
positive control. The 12-kb fragment was purified on agarose
gels prior
to transfection. FAB cells (8 × 10
4) were plated in
12-well (17-mm-diameter) tissue culture dishes.
The cells were passaged
once to 35-mm-diameter dishes before being
stained.
Cell-free supernatants were obtained from cell lysates plus
supernatants of either acutely infected HEL cells or chronically
infected H92 cells. The supernatants were filtered through
0.2-µm-pore-size
filters or centrifuged at 2,700 ×
g
for 30 min at 4°C to remove
cell debris. Virus was pelleted through a
20% sucrose cushion
containing standard buffer (SB; 100 mM NaCl, 10 mM
Tris, 1 mM
EDTA [pH 8.0], 20% sucrose) by centrifugation at
24,000 rpm in
a Beckman SW50.1 rotor for 2 h. Pellets were
resuspended in SB
with 10 mM MgCl
2 at 1/1,000 the original
supernatant volume. Samples
were divided in half to isolate RNA and to
isolate DNA in parallel.
These concentrated virus samples were then
treated with RQ-1 RNase-free
DNase (Promega, Madison, Wis.) (1 µl per
50 µl of sample volume)
at 37°C, for 1 h, before the viral
suspensions were divided into
two aliquots for extraction of RNA or
DNA.
In some experiments, HFV particles obtained from pooling lysates and
concentrated supernatants from HFV-infected HEL cells
were purified on
sucrose gradients after pelleting through sucrose.
Pellets were
resuspended in 200 µl of SB, treated with RQ-1 RNase
free DNase,
layered on top of a 5-ml 20 to 60% sucrose gradient
(in SB), and
centrifuged in a Beckman SW50.1 rotor for 2 h at
36,000 rpm.
Five-hundred-microliter fractions were collected,
and the refractive
index of each sample was measured. Ten microliters
of each fraction was
assayed for infectivity by using FAB cells.
Fractions with a density of
1.14 to 1.17 g/cm
3, and containing the peak of infectious
virus, were pooled, diluted
1:5 in SB, and centrifuged for 2 h at
24,000 rpm. The resulting
pellet was resuspended in 200 µl of SB and
used to isolate nucleic
acid after RNase or DNase treatment as
described
below.
Nucleic acid extractions from HFV particles. (i) RNA.
An
equal volume of buffered phenol, containing 4 M guanidinium
isothiocyanate, was added at a 2:1 ratio to the concentrated virus
sample. Samples were extracted twice with chloroform-isoamyl alcohol
(24:1). Nucleic acid was precipitated in the presence of 10 µg of
carrier glycogen (RNase free), and pelleted nucleic acids were
resuspended in the original sample volume in diethylpyrocarbonate (DEPC)-treated double-distilled water (ddH2O). Samples were
digested with RQ-1 RNase-free DNase (Promega) at 37°C for 1 h.
The sample was then reextracted as described above and resuspended in
the original volume of DEPC-treated ddH2O.
(ii) DNA.
Sodium dodecyl sulfate was added to the
concentrated virus sample at a final concentration of 0.5%. Samples
were extracted twice with a 24:24:1 mixture of
phenol-chloroform-isoamyl alcohol, and nucleic acids were precipitated
with ethanol. Pelleted nucleic acids were resuspended in the original
volume of ddH2O and treated with RNase A (Sigma) at 37°C
for 1 h. The sample was then reextracted as described above and
resuspended in the original volume of ddH2O.
(iii) RNA and DNA extraction from gradient-purified virions.
Particles were treated with RQ-1 RNase-free DNase as described above.
Nucleic acid was extracted from gradient-purified virus by using the
QIAmp HCV RNA isolation kit (Qiagen, Valencia, Calif.) as specified by
the manufacturer. Samples eluted by this procedure contained both RNA
and DNA. The samples were divided into two equal portions. One half was
treated with RNase to obtain DNA, and the other was treated with DNase
to obtain RNA. Both samples were then extracted with
phenol-chloroform-isoamyl alcohol as described above. The samples of
RNA or DNA were then precipitated with ethanol and resuspended in equal
volumes of DEPC-treated ddH2O.
Quantitative competitive PCR and RT-PCR.
The competitor DNAs
used were derived as follows. Gag plasmid pCR/H3RR, containing the NC
domain of gag (nucleotides [nt] 2623 to 3270), was
constructed by inserting a 647-bp PCR fragment from a previously
described gag deletion mutant cloned in the pCR II (2.1)
vector (Stratagene, La Jolla, Calif.). The mutation in pCR/H3RR is a
55-bp deletion from nt 2074 to 2129 in the NC domain of gag
(35). Plasmid pKS/dU3R, containing a portion of the U3
repeat (R) region of HFV (nt 316 to 868), was constructed by inserting
a mutant 485-bp PCR fragment (digested at unique SmaI and
HindIII sites within the primer sequences) into the
pBluescript II KS+ vector (Stratagene). The mutation in pKS/dU3R is a
67-bp deletion, created by a two-step PCR mutation, between nt 660 and 727 in the U3 and R regions of the HFV genome. RNAs for use as competitors in the RT-PCRs were transcribed from pCR/H3RR and pKS/dU3R
by using the enzymes SP6 and T7 RNA polymerase, respectively. Both RNA
and DNA were quantitated by determining the optical density at 260 nm.
PCR.
Each PCR mixture consisted of 1× PCR buffer
(Perkin-Elmer, Branchburg, N.J.), 1.5 mM MgCl2, 0.1 mM
deoxynucleoside triphosphate mix (Gibco/BRL, Grand Island, N.Y.), 1 U
of Perkin-Elmer Taq polymerase, and 4 ng of each primer.
Samples were denatured for 2 min at 95°C before thermal cycling was
done. Temperatures for denaturing, annealing, and extension were
95°C, 44°C (U3-R) or 53°C (H3RR), and 72°C, respectively, at 1 min each for 30 cycles. The final extension reaction consisted of
specific annealing at 72°C for 10 min, extension at 72°C for 1 min,
and primer extension at 72°C for 10 min. The U3-R versus HFV PCR was
done by using primers 316 (5'-CTGCCGGGATCAGAACATTGACAGA) and 868 (5'-AAGCTTCAGCGAGTAGTGAAG). The Gag versus HFV
PCR was done by using primers NC#8
(5'-ACTTCTAGACCCTCTCAAGGACCAG) and PR#2
(5'-CATGGGTACCGTTGCCCCTGAATCCCAG). (the HFV
complementary primer sequence is underlined.)
RT-PCR.
A constant amount of viral RNA and increasing
amounts of competitor RNA transcripts were combined in reverse
transcription-PCR reaction mixtures containing 2.5 U of avian
myeloblastosis virus RT (U.S. Biochemicals, Cleveland, Ohio) and 6 U of
RNase inhibitor (Boehringer Mannheim, Indianapolis, Ind.) at 42°C for
45 min. Reaction mixtures were then subjected to PCR as described above.
All PCR and RT-PCR products were electrophoresed on 2% Nusieve agarose
gels in 1× Tris-borate-EDTA
buffer.
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RESULTS |
Quantitation of RNA and DNA in particles.
Our previous studies
(34) showed that about 10 to 15% of extracellular viral
particles contain ca. 12.4 kb of double-stranded DNA as detected by
Southern blotting and electron microscopy. DNA of this size is expected
to contain the entire HFV genome. To more carefully quantitate the
amounts of RNA and DNA in HFV virions, we used quantitative competitive
PCR and RT-PCR (24). For this assay, competitor DNAs and
RNAs were prepared in vitro. These competitors are identical to the
viral sequence except for a small region between the PCR primers, which
is deleted to allow differentiation of PCR products resulting from
viral and competitor templates. When the level of DNA or RNA competitor
and the level of test nucleic acid are the same, an equivalent amount
of PCR product is seen for each.
Virus was prepared from the supernatants from chronically infected H92
cell lines or from acutely infected HEL cells. Viral
particles were
pelleted through sucrose cushions and treated with
DNase before nucleic
acids were extracted from the virions. Since
HFV-infected cells contain
a large amount of linear viral DNA,
we wanted to minimize the amount of
contaminating DNA from lysed
cells. We also used virus that had been
treated with DNase and
then further purified on sucrose density
gradients. After virions
were lysed, equal aliquots of the samples were
treated with DNase
before isolation of RNA or with RNase before
isolation of DNA.
Two different sets of PCR primers were used. One set
of primers
amplifies sequences between the U3 and R regions
of the viral
LTRs. This set of LTR primers is expected to hybridize to
two
locations in two-LTR-containing viral DNA but only once in viral
RNA (Fig.
1A). The second set of primers
hybridizes to the 3'
end of the viral
gag gene (Fig.
1B).
Both sets of primers will
hybridize only to DNA which has been extended
after the first
reverse transcriptase jump and do not measure
strong-stop DNA
created after extension from the primer binding site to
the 5'
end of the RNA.
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FIG. 1.
Quantitative competitive RT-PCR and PCR of foamy virus
particle nucleic acids. (A) Map of the ends of two-LTR-containing
linear HFV DNA showing the location of the U3-R primers. The boxes
represent the ends of the viral DNA, with the ends of the viral RNA
indicated above by a thick solid line. The lines flanked by arrowheads
below the boxes indicate the locations of the U3-R primers used.  67,
deletion in the competitor nucleic acids. Abbreviations: U5, unique 5'
region; PBS, primer binding site. (B) Map of the 5' end of the HFV DNA
showing the location of the gag primers. The location of the
primer is indicated by the line flanked by arrowheads. 55, deletion
in the competitor nucleic acid, from the H3RR mutant. (C) RT-PCRs
(left) and PCRs (right) using the U3-R primers. Viral nucleic acids
were resuspended after ethanol precipitation so that each 1-µl
aliquot represents 1 ml of original cell supernatant. The amount of
competitor RNA (RT-PCR) or DNA (PCR) used is shown above each lane.
Lanes 1 contain competitor only in the reaction mix, and lanes 7 show
reactions done without competitor. (D) RT-PCRs (left) and PCRs (right)
using the gag primers. Nucleic acids and designations are as
for panel C, except that no competitor is in lane 9 for RT-PCRs or lane
7 for PCRs. The competitor-only lanes for the RT-PCR reaction are not
shown.
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Using deleted competitor RNAs or DNAs, we were able to directly assess
the relative concentrations of RNA and DNA in extracellular
particles.
We initially did titrations over a wide range of competitor
RNA or DNA
concentrations and then focused on the concentrations
around the
equivalence point in the PCRs. Experiments were done
with virions from
the supernatants of both persistently infected
H92 cultures (Fig.
1)
and from acutely infected HEL cultures (data
not shown), with the same
results. Figure
1 shows the results
of one set of experiments. We
compared the competition levels
for RNA and DNA by using the U3-R
primers and found that there
were equivalent competition levels for the
RNA (RT-PCR) (Fig.
1C, left panel) and DNA (PCR) (Fig.
1C, right panel)
in the extracellular
particles. In both cases, there was approximately
50% competition
with 2 ng of competitor. Since full-length viral DNA
contains
two copies of the U3-R region and the RNA contains only one
copy,
this indicates that there is about twice as much RNA containing
U3-R sequences as there is DNA. Using the primers which hybridize
to
the 3' end of
gag, we found equivalent signals from viral
nucleic
acid and competitor with 0.2 ng of competitor in the RT-PCRs
(Fig.
1D, left panels) and 0.1 ng of competitor in the PCRs (Fig.
1D,
right panel). This again indicates that there is about a 2:1 ratio
of
RNA to DNA in extracellular virions. This experiment was performed
seven times, with variations in the level of competition. When
the H3RR
competitor was used, the ratio of RNA to DNA varied from
2:1 to 10:1,
with an average of 4.6:1. We also did the competition
experiment with
virus that had been purified over a 20 to 60%
sucrose gradient, from
which gradient fractions of 1.4 to 1.7
g/ml were pooled. When fractions
containing the peak of infectivity
were pooled and repelleted, we found
that we had an approximately
30-fold loss of both RNA and DNA. However,
the ratio of RNA to
DNA in the sucrose gradient virus did not vary from
that in the
nongradient purified virus. We found that again there was a
ratio
of RNA to DNA of 4:1 in the gradient-purified particles.
Therefore,
we conclude that about 20% of the particles released from
HFV-infected
cells contain DNA intact enough to have 3'
gag sequences.
Infectivity of virion DNA.
To determine whether any of the
virion-associated DNA is full length and capable of directing
the synthesis of infectious virions, we prepared DNA from
extracellular particles. These particles were treated with DNase before
nucleic extraction was performed to minimize contamination with
cellular DNA. DNA was then introduced into FAB cells by using
Lipofectamine, or Lipofectamine Plus reagent, which we have found to be
the most efficient way of transfecting these cells. Although some of
the virion DNA appeared to be of proviral length by Southern blotting
(34), it is possible that the DNA contains nicks or gaps, as
in the case of HBV genomes (reviewed in reference
7). As a control, we used full-length two-LTR-containing linear DNA excised from the pHFV13 molecular clone.
Rather than staining the cells directly, transfected cells were
trypsinized and replated on larger plates to allow cell growth and
viral spread and thus to amplify the signal. The results are given in
Table 1. We found that we could detect
infectivity in 1 ng of plasmid DNA and in 50 ng of virion DNA. Smaller
amounts of either plasmid or virion DNA were negative for infectivity. Controls without added DNA or with 1,000 ng of irrelevant pCITE plasmid
DNA yielded no blue cells. All of the positive plates showed clusters
of cells staining positive for -Gal (Fig.
2). At the higher levels of DNA,
transfection with either control plasmid DNA (Fig. 2A) or virion DNA
(Fig. 2B) led to formation of giant multinucleate syncytia and massive
cytopathic effects. Even at low concentrations of virion DNA (Fig. 2C
and D), many -Gal-positive cells were seen in clusters, indicating
the spread of infectious virus. Thus, virion DNA is about 1/50 as
infectious as plasmid DNA. We have not been able to repeat the
infectivity assays with sucrose gradient-purified DNA because of the
ca. 30-fold loss resulting from such purification. It is not feasible
to grow and purify enough virus to do this experiment.
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FIG. 2.
Photographs of FAB cells transfected with plasmid or
virion DNA. Plates obtained as described for experiment B in Table 1
were photographed at ×250 magnification after staining with X-Gal.
Cells were transfected with 1 ng of linearized pHFV13 DNA (A), 1 µg
of virion DNA (B), 100 ng of virion DNA (C), and 50 ng of virion DNA
(D).
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Effect of inhibitors on viral infection.
In the case of
conventional retroviruses, viral particles contain essentially only RNA
genomes, and reverse transcription is required as an early step after
infection of new cells. Thus, infection is sensitive to inhibitors of
reverse transcription, such as AZT. However, once cells are infected,
reverse transcription is not required for production of infectious
particles, so adding inhibitors to infected cells does not abrogate the
ability to produce infectious virus (2) (Fig.
3, left). We reasoned that if the
functional foamy virus genome was DNA rather than RNA, then inhibitor
studies should give the opposite results (Fig. 3, right). That is,
reverse transcription should be a late event in viral production and
virions should be able to infect cells that had been pretreated with
inhibitors. Conversely, virions produced from inhibitor-treated cells
should not be infectious.
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FIG. 3.
Diagram of the strategy of, and expected results from,
AZT experiments. The left side of the diagram shows the expected
results if the genome is RNA as in conventional retroviruses; the right
side shows the predicted results for a DNA-containing virus. Symbols:
×, virus cannot replicate;  , virus can replicate.
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We tried a series of RT inhibitors for their effects on viral infection
and infectious virus production. We could not show
any inhibition with
the drug lamivudine (3TC) at concentrations
known to inhibit HIV (data
not shown). This is not surprising,
given that the catalytic site
sequence YMDD, which is thought
to be important for lamivudine
inhibition (
28), is YVDD in HFV
RT. Contrary to the findings
in a published report (
27), we
were unable to demonstrate
any effect on HFV inhibition with the
inhibitor ddI, even at
concentrations as high as 10 µM (data not
shown). This may also be
caused by the sequence being YVDD rather
than YMDD (
10).
However, we did find that AZT was a potent inhibitor
of HFV production,
and detailed analyses were done with this drug.
We used a high
concentration of AZT in our studies, generally
100 µM, to ensure that
any lack of an effect on viral infectivity
was significant. This
concentration is not toxic for FAB or H92
cells (data not
shown).
Next, we analyzed the ability of AZT to prevent HFV13 infection of FAB
cells which had been pretreated with AZT (Table
2).
We found that in over a dozen
different experiments, AZT had only
modest effects on the ability of
untreated virus to infect the
cells, which had been pretreated with AZT
for 4 h. In these experiments,
viral infection was measured
directly by staining the FAB cells
with chromophore at different times
after infection. The decreases
ranged from 50- to 70-fold in one
experiment to 3- to 7-fold in
the majority of the experiments. The
average difference in infection
between untreated and cells treated
with 100 µM AZT in these experiments
was 0.17 at 48 h after
infection. We examined the effect of AZT
on a control retroviral
vector, MLV encoding
-galactosidase,
which was pseudotyped with VSV
G protein. This allowed us to compare
the effects of inhibitor in the
hamster-derived FAB cells, as
well as mouse NIH cells (Table
3). We found that AZT decreased
infectivity, as measured by chromophore staining of the cells,
by 300- to 1,000-fold, as was observed in similar experiments
which measured
the effects of AZT on HIV infectivity (
4). In
this
experiment, the effect of AZT on HFV was about a 10-fold
decrease in
infectivity.
In several experiments, a time course of infection showed little change
in the ratio of infectivity from 1 to 3 days after
infection. At 3 days
after infection, the control cells exhibited
abundant syncytia
formation, indicative of a spreading infection,
while the AZT cultures
did not have any syncytia. As discussed
below, this is because AZT
prevents formation of infectious particles.
HFV derived from another
infectious molecular clone, PHFV-2 (
11),
yielded similar
results (data not
shown).
We next examined the ability of FAB cells that were treated with AZT,
and then infected, to produce infectious virus when
assayed on
untreated cells (Table
4). We found that
in contrast
to the results shown in Table
2, AZT had a marked effect on
the
inhibition of infectious virus production, which ranged from
100-fold
to greater than 3,000-fold, in different assays. This is again
different from the results with conventional retroviruses. We
also
examined the effect of AZT on infectious virus production
from
persistently infected H92 cell clones (Table
5). We grew
these cells in 100 µM AZT
and determined the titer of the virus
after 6 days to 8 weeks. Again we
saw variations in the assays,
but in all cases, inhibition of
infectious virus production was
greater than 150-fold. Since the
control MLV
-Gal vector cannot
replicate, we were unable to use it
in this assay.
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DISCUSSION |
Retroviruses and pararetroviruses share a requirement for RT
activity in their life cycles. However, the timing of reverse transcription is very different for these two viral groups.
Conventional retroviruses such as HIV have RNA genomes, and reverse
transcription occurs primarily after infection of cells. Linear viral
DNA is found in the cytoplasm and is transported into the nucleus,
where it is integrated into the genome as proviral DNA. Proviral DNA is
present for the life of the infected cell and is the template for viral
transcription and translation. In contrast, virus budded from
hepadnavirus (pararetrovirus)-infected cells contains gapped circular DNA which is converted to covalently closed circular DNA
(cccDNA) after infection, a step which is thought to require RT
activity. This cccDNA does not usually integrate into the genome. Infection is maintained by small numbers of cccDNAs per cell, since the
infected cell is a mature hepatocyte which does not normally divide
(30).
The genomes of foamy viruses clearly place them in the retrovirus
family, yet they share more features with the hepadnavirus group of
pararetroviruses than other retroviruses do. The work presented here
lends credence to the idea that the foamy virus group is unique among
the retroviruses, and its replication strategy has evolved in a
different direction more closely related to that of the hepadnaviruses.
Thus, the most recent classification of retroviruses into seven genera
(mammalian type B, mammalian type C, avian type C, type D, bovine
leukemia virus-human T-cell leukemia virus, Lentivirus, and
Spumavirus) is somewhat misleading, with Spumavirus being much more distantly related to the other
six. While the nature of the genome of other members of the
Spumavirus genus has not been characterized, both feline and
bovine foamy viruses also synthesize RT from a spliced mRNA rather than
as a Gag-Pol fusion protein (5, 12) and therefore are likely to activate RT at a similar stage in viral maturation as HFV. However,
similar analyses of the virion genomes have not as yet been done.
Our data show that mature extracellular particles contain apparently
full-length DNA molecules of about 12.4 kb in length that probably
contain two LTRs. We had previously suggested that about 10 to 15% of
the particles contain such DNA and that there were sufficient
DNA-containing particles to account for viral infectivity
(34). Using a more quantitative assay, we confirmed that
about one-fifth of extracellular particles contain DNA containing U3-R
and gag sequences. This might appear to be a large amount of
RNA in a virus with a DNA genome. Much of this RNA could be due to
reverse transcription which is aborted at an early stage. There is no
information as to how efficient reverse transcription is after
infection of cells by conventional retroviruses, since it is not easy
to purify uncoated cytoplasmic particles after reverse transcription,
but before nuclear transport, and examine them for nucleic acid
content. Clearly, an integrated provirus does not result from every
infecting retroviral particle, and it may be that reverse transcription
occurring early in infection is also quite inefficient. We have no
further information about the structure of the HFV DNA and do not know
whether the DNA molecules contain nicks. However, a fraction of the
virion DNA can lead to production of viral particles when transfected
into susceptible tissue culture cells. The 50-fold-lower infectivity
compared to that of plasmid-derived DNA could indicate that many of the
packaged DNA molecules contain nicks or gaps which need to be repaired prior to integration. In fact, gapped DNA intermediates have been reported in HFV-infected cells, probably as a result of positive-strand DNA initiation at more than one site (15, 31). If HFV nicks are normally repaired by RT, this could account for the low infectivity seen in our assay. It is not known whether DNA extracted from extracellular hepadnavirus particles is infectious because of the lack
of suitable tissue culture infectivity assays.
These studies show that virion DNA is abundant and can lead to the
production of infectious virus. The result of experiments with AZT, a
potent inhibitor of RT, are consistent with DNA as the major functional
genomic molecule. When AZT is added to chronically or lytically
infected cells, little or no infectious virus is produced. If RNA can
serve as a viral genome as well as DNA, then one would expect only a
modest effect on viral infectivity since at least 60% of the virions
contain RNA. It should be noted that there are no published reports
showing the size of the HFV-packaged RNA, and it is possible that a
large fraction of the RNA is less than full length. The results of
pretreatment of cells with AZT show that wild-type levels of virus are
never produced, and the highest level of infection we have seen is 30 to 40% of that in untreated controls. In some experiments, the level
of virus after AZT treatment is much lower (Table 2). This could
indicate that upon infection, limited reverse transcription is needed
to produce DNA that is competent for integration. The fact that virus
produced from AZT-treated cells has little or no infectivity (Table 4) is also inconsistent with a major role for RNA genomes in infectivity. However, AZT could be having some other effect on viral production. We
have looked at viral protein in the supernatants of AZT-treated or
untreated HFV-infected H92 clones and have not found any differences, even after months of treatment (6a). However, it is not
possible to absolutely rule out a role for RNA in viral infection with the present data. While this manuscript was being prepared, similar, but less extensive, results with AZT treatment of HFV-infected cells
were reported (20). We have not as yet found another
inhibitor of HIV RT which prevents HFV replication. This may be due to
the differences in the sequence at the site sensitive to HIV RT
inhibitors. HFV could prove useful as a screen of RT inhibitors for
their effects on a virus which efficiently infects human cells,
including T cells and macrophages, and which naturally lacks the
methionine in the YMDD motif as do a majority of AZT-resistant HIV
particles (28).
Foamy viruses differ from conventional retroviruses in both the nucleic
acid used as a genome and their mode of expression of the Pol protein,
and these could be intimately related. In conventional retroviruses,
Pol is expressed as a Gag-Pol fusion protein, and activation of
protease (PR) and cleavage of Pol from the complex are relatively late
events in the viral life cycle, occurring during or after budding
(8, 21). In general, Gag-Pol proteins do not exhibit full RT
activity (9). In the case of HFV, the primary Pol
translation product is a PR-RT-integrase (IN) protein (23,
34). HFV PR has limited activity in the viral life cycle. Only
one cleavage in the Gag protein, near the C terminus, is seen in mature
virions (14). IN is efficiently cleaved from PR-RT-IN, and a
PR-RT protein can readily be detected in infected cells. This protein
is probably localized in intracellular virions, which are extremely
abundant in infected cells and highly infectious (37). It is
not yet known whether PR and RT are further cleaved from PR-RT or
whether this fusion protein contains both activities. It is possible
that premature reverse transcription and concomitant accumulation of
DNA in released particles occur in HFV infection because the lack of
Gag domains on the Pol protein allows early activation of the PR-RT-IN
protein during viral assembly.
Foamy virus-infected cells contain many hundreds of copies of viral DNA
(6a, 29). By using the retroviral paradigm, it has been
assumed that this DNA is cytoplasmic linear DNA, as seen after acute
infection by HIV and other retroviruses (22, 26). However,
most HFV does not bud from tissue culture cells. Since budded virions
contain large amounts of DNA, it is reasonable to assume that the
cell-associated HFV DNA is actually in intracellular viral particles,
which are present in large numbers both at intracellular membranes and
in the cytoplasm (3). This intracellular pool of particles
may be analogous to the cccDNA of hepadnaviruses and provides a way to
recycle DNA into the nucleus for integration. Although very little is
known about how foamy viruses are maintained in vivo for long periods
of time, pools of intracellular DNA containing virions which escape
immune surveillance could be a reservoir for persistent infection in
infected animals.
| |
ACKNOWLEDGMENTS |
Shuyuarn F. Yu and Mark D. Sullivan contributed equally to the work.
We thank Mike Emerman, Julie Overbaugh, and other members of the
Thursday virology research group for their insightful critiques of this work.
This work was funded by grants CA 18282 and HL 53763 to M.L.L.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Div. of Basic
Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., A3-015, P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-4442. Fax: (206) 667-5939. E-mail: mlinial{at}fhcrc.org.
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Journal of Virology, February 1999, p. 1565-1572, Vol. 73, No. 2
0022-538X/99/$04.00+0
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Top
Abstract
Introduction
