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RETROTRANSPOSONS AS MODELS FOR THE
REPLICATION OF RETROVIRUSES
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Henry
L. Levin, PhD, Head, Section on
Eukaryotic Transposable Elements Angela
Atwood-Moore, BA, Senior Research
Assistant Tracy
Ripmaster, PhD, Research Assistant Pratiti
Das, PhD, Visiting Fellow Hirotaka
Ebina, PhD, Visiting Fellow Young-Eun
Leem, PhD, Visiting Fellow Min-Kyeong
Kim, PhD, Postdoctoral Fellow Kenechi
Ejebe, BA, Postbaccalaureate Fellow Marc
Heincelman, BA, Postbaccalaureate
Fellow Felice
Kelly, BA, Postbaccalaureate Fellow Julie
McClure, BA, Postbaccalaureate Fellow Jay
Myung, BA, Postbaccalaureate Fellow Amnon Hizi, PhD, ORISE Fellowa |
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Diseases
caused by retroviruses such as HIV-1 and the adaptation of retroviruses for
use in gene therapy have intensified the need to understand how these viruses
replicate. Our primary objectives are to understand how reverse transcription
of viral mRNA occurs and how the cDNA products are integrated into the genome
of infected cells. Owing to their similarity to retroviruses,
LTR-retrotransposons are important models for retrovirus replication. The
retrotransposon under study in our laboratory is the Tf1 element of the
fission yeast Schizosaccharomyces pombe. Tf1 efficiently
reverse-transcribes its mRNA, and its integrase (IN) inserts the cDNA into
the genome of S. pombe. The techniques of yeast genetics greatly
facilitate our studies of reverse transcription and integration. To monitor
the effect of various mutations on transposition, we express copies of Tf1.
Not only does this approach allow us to identify features of the transposon
critical for activity, but it also enables us to identify host genes required
for transposition. Large-scale screens of mutant strains have identified
novel features of reverse transcriptase (RT) and have led to the discovery of
new host genes necessary for transposition. The ease of culturing yeast also
allows us to investigate biochemically the mechanisms we identify by
genetics. Integration
of Tf1 specifically at Pol II promoters Kelly, Das, Ebina,
Myung, Levin The
integration of HIV-1 cDNA shows a significant preference for actively
transcribed genes. Similarly, the insertion of murine leukemia virus shows a
preference for sites within 5kb of pol II–initiated transcription.
Little is known about the interaction of the viruses with the structures of
chromatin and the recognition of target sites. The complete DNA sequence of
the genome of S. pombe provided the opportunity to investigate the
entire complement of transposable elements (TEs) and their chromosomal
distribution. Our analysis identified 186 pre-existing insertions of Tf
transposons. To determine whether preferences for integration sites existed
during the insertion of the 186 Tf sequences, we compared the sites’
locations to the positions of all 4,984 predicted ORFs (open reading frames)
of S. pombe. We found that all insertions were located exclusively in
intergenic regions of the genome that contained pol II promoters. In
addition, the LTRs (long-term repeats) were clustered within 300 nucleotides
of the 5´ end of the ORFs. Such specific positions could be the result of (1)
selective pressures, either positive or negative, that favor populations of S.
pombe with each of the patterns observed or (2) biochemical mechanisms of
integration. Each of the biases in the position of Tf sequences described
above were highly similar in pattern and magnitude to the positions of insertions
resulting from the induction of Tf1 transposition under laboratory
conditions. Such extensive similarities argue strongly that the biases in the
position of Tf sequences result from biochemical preferences of integration
for specific sites. Furthermore, the data indicate that Tf elements recognize
and insert upstream of RNA polymerase II promoters.
This year,
by creating an integration assay for specific target plasmids, we explored
the ability of Tf1 to recognize pol II promoters. We generated a plasmid with
the ade6 gene of S. pombe and included it in a strain induced
for Tf1 transposition. We isolated and analyzed 50 separate insertions into
the target plasmid. Ninety-five percent of the inserts occurred within a
160-nucleotide window of the promoter of ade6 (see Figure 13.1). The
target assay clearly reproduced the promoter preference that we observed with
insertions into genomic sites. The insertions within the 160-nucleotide
window exhibited a strong pattern of periodicity. Four narrow clusters of
inserts were spaced approximately 30 bp apart (see Figure 13.2). The window
of 160 nucleotides corresponded to the amount of DNA wrapped around a single
nucleosome; furthermore, the 30 bp pattern corresponded to the amount of DNA
bound to the histone fold pairs within a single nucleosome.
The
IN of Tf1 contains Zn-finger and catalytic domains similar to those of
retroviral INs. Unlike other INs, the Tf1 IN possesses a chromodomain at its
C-terminus. Chromodomains have been found to bind directly to histone H3 in
specific nucleosomes, suggesting that Tf1 integration is mediated by an
interaction between IN and specific nucleosomes at pol II promoters.
Consistent with this model, we found that IN purified from bacteria bound
specifically to histone H3 but not to H2a, H2b, or H4. We observed that the
insertions we isolated in the promoter of ade6 correspond to a
location where, according to published data, a positioned nucleosome exists,
constituting further evidence that Tf1 recognizes histones. We are currently
testing whether, under the conditions of our integration assay, a positioned
nucleosome exists in our target plasmid. Bowen N, Jordan I, Epstein J, Wood V, Levin H. Retrotransposons
and their recognition of pol II promoters: a comprehensive survey of the
transposable elements derived from the complete genome sequence of Schizosaccharomyces
pombe. Genome Res 2003;13:1984-1997. Kelly F, Levin HL. The evolution of retrotransposons in Schizosaccharomyces
pombe, retrotransposible elements and genome evolution. In: Volff J, ed. Cytogenetics
and Genome Research. Würzburg: in press. Levin H. The retrotransposons of Schizosaccharomyces pombe.
In: The molecular biology of Schizosaccharomyces pombe. Singleton T, Levin H. An LTR-retrotransposon of fission yeast
has strong preferences for specific sites of insertion. Eukaryotic Cell
2002;1:44-55. The
chromodomain of Tf1 IN, a negative regulator of integration Hizi, Levin; in
collaboration with Davies To
study the function of the chromodomain of Tf1 IN, we developed biochemical
assays to measure the activities of the protein. We attached a six-his tag to
the N-terminus of Tf1 IN and purified the protein from E. coli, using
Ni resin. Even though the INs of retroviruses, including that of HIV-1, are
extremely insoluble and difficult to concentrate, we were surprised to find
that the full-length IN of Tf1 was easy to purify and could be readily
isolated in high concentrations. The INs of retroviruses possess processing
activity that removes two terminal nucleotides from the 3´ ends of the cDNA.
Using oligonucleotides that mimic the ends of the cDNA, we assayed the Tf1 IN
for processing activity. The IN had strong processing activity that removed
from two to five additional nucleotides from the 3´ end of the
oligonucleotides in vitro, suggesting that the multiple nucleotides
present at both ends of the cDNA could be removed by IN in vivo. This
would allow integration of the most prevalent cDNA species we detected. We
also found that the Tf1 IN exhibits strong IN activity, as indicated by the
ability of the enzyme to insert oligonucleotides into each other. The
resulting products were substantially larger than the original oligonucleotides.
Oligonucleotides that mimicked the donor cDNA were altered to test whether
Tf1 IN required specific sequences at the 3´ ends. As is the case with other
INs, Tf1 IN had a strong requirement for the dinucleotide CA at the 3´ ends.
The results of the processing and integration assays demonstrate that the IN
of Tf1 has the same activities as retroviral INs and is therefore an
excellent model for the IN of HIV-1. In addition, the high solubility of the
Tf1 IN suggests that the protein may form crystals, allowing the first
high-resolution structure of an intact IN to be determined and thus
significantly expanding our understanding of retrovirus integration. We are
pursuing this possibility in collaboration with the laboratory of David
Davies. To explore
the function of the chromodomain, we expressed in E. coli a Tf1 IN
that lacked the chromodomain. We assayed the chromo-minus IN (CHˉ) for
integrase activities by using the artificial substrates described above. CHˉ
possessed strong activities in both the processing and integration reactions.
We were surprised to find that the enzyme lacking the chromodomain was about
10 times more active than the intact IN. We also tested whether the
chromodomain contributed to the recognition of the terminal dinucleotide at
the 3´ end of the donor DNA. The CH protein exhibited a surprising relaxation
of the sequence requirement at the ends of the donor DNA. Taken together, the
results indicate that the chromodomain functions as a negative regulator of
integration and as a specificity factor for the donor DNA. We are currently
testing the possibility that the chromodomain inhibits integration until it
identifies a specific nucleosome at a pol II promoter. The chromodomain then
binds to H3 of that nucleosome, which alters the conformation of IN and thus
stimulates integration activity. The
primer grip in the RNase H of RT, a conserved domain that contributes
specifically to removal of the plus strand primer from the 5´ end of the cDNA
Atwood-Moore, Ejebe,
Levin; in collaboration with LeGrice Reverse
transcription of retroviruses and LTR-retrotransposons is a complex sequence
of reactions that produces several critical intermediate products, including
the initial minus strand product of cDNA called a strong stop, the extended
minus strand, the plus strand primer (PPT), the plus strand strong stop, and,
ultimately, the full-length double-stranded cDNA. The synthesis of these
intermediates requires both the DNA polymerization and RNase H activities of
RT. The RNase H domain must degrade the RNA once it is used as template and
remove the PPT once it has primed plus-strand synthesis. Although much is
known about the amino acids that catalyze the DNA synthesis and RNA
degradation, less is known about which residues and structures are required
for the recognition and removal of the PPT. Last
year, we identified residues of Tf1 RT that mediate the removal of the PPT
from the ends of the cDNA. We used random mutagenesis of RT and two genetic
assays to identify mutations that inhibited integration but not reverse
transcription. Our experiments focused on a cluster of mutations in RNase H
that included a region with five mutations in a five–amino acid
segment. A crystallographic study of HIV RT demonstrated the significance of
the domain and showed it as a “primer grip,” which interacts
directly with the nucleotides of the PPT adjacent to the position that is
cleaved by RNase H, thus creating and then removing the PPT. Both the
position and sequence of the primer grip residues in HIV corresponded to the
cluster of mutations we identified in the RNase H of Tf1. These observations
led us to propose that the mutations in the RNase H of Tf1 that result in
full-length cDNA are defective for transposition because the recognition of
the PPT is impaired. A defect of a few nucleotides in the cleavage of the PPT
from the 5´ end of the plus strand would be templated into altered sequences
at the 3´ end of the minus strand, which would have a drastic impact on the
ability of IN to catalyze strand transfer. A test of our hypothesis revealed
that the cluster of mutants in RNase H did inhibit the removal of the PPT
from the cDNA. The
residues in the RNase H necessary for the removal of the PPT are within a
conserved region of RT, suggesting that the function of the domain may also
be conserved. To test such a possibility, we created corresponding mutations
in the RNase H of Ty3, an LTR retrotransposon closely related to Tf1. Work
concluded this year revealed that several mutations in the RNase H of Ty3
exhibited the same properties as those isolated in Tf1; the mutations in Ty3
significantly reduced transposition activity without inhibiting expression of
RT or synthesis of cDNA. Given that such mutations produced normal levels of
full-length cDNA, we concluded that the defects in RNase H alter the removal
of the primers from the cDNA, thus inhibiting the ability of IN to complete
integration. We selected Ty3 as the transposon for these experiments because
Stuart LeGrice’s laboratory had developed methods to express Ty3 RT and
measure the activities of its RNase H. In a collaboration, the LeGrice
laboratory expressed the Ty3 RTs in E. coli and tested them for RNase
H activities. Assays with model substrates revealed that two RTs exhibited
defects in removal of the PPT primer, but not in other RNase H activities.
These biochemical experiments support the results of the genetic experiments
and indicate that the primer grip of RNase H plays a specific role in
removing the PPT from the 5´ ends of the cDNA. Regulation
of nuclear localization and particle assembly by amino acids in Gag of the
LTR-retrotransposon Tf1 Kim, Levin The
IN and cDNA of retroviruses form a preintegration complex (PIC) that must
access the nucleus to perform integration. Because HIV-1 infects nondividing
cells, the PIC must enter the nucleus through the nuclear pore complex (NPC).
The matrix, virus protein R, IN, and DNA flap structure of the viral cDNA are
among the components that have nuclear-localizing activity and may contribute
to the nuclear import of the PIC. In addition to its ability to traffic
through the NPC of nondividing cells, the PIC of HIV-1 can be imported into
the nucleus of dividing cells while the nuclear envelope remains intact,
evidence underscoring the possibility that mechanisms of nuclear import may
be important for the propagation of other retroviruses. In an
effort to model the import of HIV-1 into the nucleus, we examined the nuclear
import of Tf1 in S. pombe. The Gag and cDNA of Tf1 enter the nucleus
only after cells reach the stationary phase of growth. Previous studies
identified a nuclear localization signal (NLS) in the N-terminus of Gag, a
signal that is required for transposition. Mutations in the NLS cause a
severe defect in the nuclear localization of Gag and the cDNA. In separate
experiments, we found that a factor of the nuclear pore, Nup124p, had a
specific activity required for nuclear import of Tf1. Mutations in nup124
cause a significant defect in the import of Tf1 Gag and, surprisingly, do not
reduce the import of other proteins. The results of two-hybrid analyses and
precipitation studies revealed an interaction between the N-terminus of
Nup124p and the Gag of Tf1. We proposed that the binding of Gag to Nup124p
could mediate the nuclear import of Tf1. We
further explored the function of Nup124p in the import of Tf1 Gag by studying
the import of large proteins consisting of sections of Gag fused to GFP and
lacZ. Interestingly, Nup124p was required for import of the first 50 amino
acids of Gag fused to GFP-lacZ. When specific segments of Gag were removed
from the fusion, the requirement for Nup124p was lost. The requirement for
Nup124p was mapped to residues 10 through 30 of Gag. To understand how the
Gag of Tf1 is imported into the nucleus and the role of Nup124p in this
process, we introduced five independent mutations in Gag residues 10 through
30. The transposition activities of the Tf1 elements containing the five
alanine mutations A1, A2, A3, A4, and A5 were significantly less than that of
the wild-type Tf1, indicating that the region of Gag from residues 11 to 30
is critical for transposition. The
localization of Gag by indirect immunofluorescence revealed that the Gags of
mutants A1, A2, and A3 were not imported into the nucleus. These residues are
adjacent to the NLS and may contribute to its activity. It was interesting
that the localization of Gag for A4 and A5 occurred in the nucleus at both
the log and stationary phase of cell growth and, in particular, that the
import of Gag with the A5 mutation did not depend on Nup124p. The reduced
localization of Gag in the nucleus caused by mutations A1, A2, and A3 and the
premature localization of Gag in the nucleus of log-phase cells caused by
mutations A4 and A5 may be attributable to changes in the structure of the
virus-like particles (VLPs). The assembly of Gag, RT, IN, and cDNA into VLPs
can be detected as large macromolecular complexes. By subjecting cell
extracts to gradient sedimentation and electron microscopy, we tested whether
the alanine mutations altered the formation of the particles. The
sedimentation of Gag-A4 and Gag-A5 was significantly reduced compared with
wild-type Gag. These results and the electron micrographs indicate that the
A4 and A5 mutations significantly disrupt the structure of Tf1 particles.
Therefore, the ability of Gag to enter the nucleus corresponds to the loss in
particle structure, suggesting that, at least in stationary-phase cells, the
import of Tf1 into the nucleus is impeded by its particle structure and that
Nup124p is required to overcome this block. Silverstein R, Richardson B, Levin H, Allshire R, Ekwall K. A
new role for the transcriptional co-repressor SIN3; regulation of
centromeres. Curr Biol 2003;13:68-72. Teysset L, Dang V, Kim M, Levin H. An LTR-retrotransposon of Schizosaccharomyces
pombe expresses a Gag-like protein that assembles into virus-like
particles and mediates reverse transcription. J Virol 2003;77:5451-5463. aTel COLLABORATORS
David Davies, PhD, Laboratory of Molecular
Biology, NIDDK, Yehuda Goldgur, PhD, Stuart LeGrice, PhD, HIV Drug Resistance
Program, NCI, Frederick, MD
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