Previous Article | Next Article 
Journal of Virology, April 1999, p. 3477-3483, Vol. 73, No. 4
Department of Molecular Genetics, Albert
Einstein College of Medicine, Bronx, New York 10461
Received 11 August 1998/Accepted 19 December 1998
A stem-loop structure at the 5' end of the R region of the long
terminal repeat (LTR) of the murine leukemia virus SL3 and other type C
mammalian retroviruses is important for maximum levels of expression of
a reporter gene under the control of the viral LTR. This element,
termed the R region stem-loop (RSL), has a small effect on
transcriptional initiation and no effect on RNA polymerase
processivity. Its major effect is on posttranscriptional processing of
LTR-driven transcripts. Here we tested whether the RSL affected the
production of RNAs from a full-length SL3 genome. Mutation of the RSL
in the 5' LTR of SL3 reduced the cytoplasmic levels of full-length
viral transcripts but not those of spliced, env mRNA
transcripts. Thus, the RSL specifically affected the cytoplasmic levels
of the unspliced viral RNA. To test further whether the effect was
specific for unspliced transcripts, a system was devised in which the
expression of a reporter gene under the control of the viral LTR was
tested in the presence or absence of an intron. Mutation of the RSL
resulted in only about a twofold decline in the level of reporter gene
expression when the transcripts contained an intron. However, when the
intron was removed, mutation of the RSL reduced expression of the
reporter gene about 10- to 60-fold in various cell lines. The secondary
structure of the RSL was essential for its activity on the intronless
transcript. Thus, the RSL appears to be important for the cytoplasmic
accumulation of unspliced viral RNA and unspliced RNA from chimeric
transcription units under the control of the viral LTR.
In addition to their role in reverse
transcriptase jumping, sequences that are located within the R regions
of the long terminal repeats (LTRs) of retroviruses affect the
production of viral RNA transcripts. The steps in viral RNA production
that are affected vary among different retroviruses. The best studied
example is the TAR element of human immunodeficiency virus type 1 (HIV-1) and related lentiviruses. The virally encoded Tat protein binds to the TAR element in nascent transcripts, resulting in the increased processivity of RNA polymerase (4, 5, 13, 14, 38, 41, 42, 47, 59,
62, 64, 69, 70). Transcription of other retroviruses, including
human T-cell leukemia virus, bovine leukemia virus, avian
reticuloendotheliosis virus, murine leukemia viruses (MuLVs), and mouse
mammary tumor virus (MMTV), is also influenced by elements within LTR R
regions (15, 19, 31, 36, 37, 40, 50, 53, 56, 58). The steps
in the production of viral RNAs that are affected vary among the different retroviruses.
We previously showed that R region sequences of MuLV SL3 affected
expression of a reporter gene under the control of a viral LTR
(15, 16). R region sequences from Moloney MuLV or feline leukemia virus could substitute for the SL3 element. Thus, the R region
element appears to be a general feature of the mammalian type C genus
of retroviruses (16). The crucial sequences were mapped to
the first 28 nucleotides of the 68-nucleotide R region of SL3. These
sequences were predicted to fold into a stem-loop structure of modest
stability. The predicted Primer extension analysis indicated that the RSL affected the levels of
cytoplasmic RNA. Nuclear run-on assays indicated that deletion of the
RSL had a small effect on transcriptional initiation and no effect on
RNA polymerase processivity (15, 16). Thus, the main effect
of the RSL was on one or more steps that occurred after the template
was transcribed by RNA polymerase. This implied that the main function
of the RSL was in RNA processing.
Mutations of the RSL in the full-length MuLV genome.
The
studies here were undertaken to ask whether the RSL was important for
transcription of the full-length viral genome. Previous studies of the
effects of the SL3 R region were performed using plasmids that
contained a reporter gene under the control of the viral LTR (15,
16). To test the importance of the RSL in the full-length viral
genome, a deletion of the RSL was made in the 5' LTR of a plasmid clone
of the full-length genome (Fig. 1A). The
deletion encompassed 28 nucleotides and removed all but the first three
nucleotides of the stem-loop structure (Fig.
2A). This deletion was identical to that
used in earlier studies with reporter plasmids (15, 16). The
experimental design was to transfect the clones of the wild-type and
mutated viruses into cultured cells and examine the viral transcripts
present in cytoplasmic RNA 48 h later. However, since only the R
region in the 5' LTR was mutated, the intact R region in the 3' LTR
would be capable of reverting the mutation during a single round of
viral replication (44). Any jump by reverse transcriptase
(RT) during minus-strand synthesis that occurred prior to reaching the
deletion in the 5' R region of the RNA template would result in the
formation of reversions in the progeny proviruses. Since we were
concerned that this would happen within 48 h in a substantial
number of genomes, we felt that it was essential to block the ability
of the virus to undergo any replication. One means to accomplish this
was to use human cells that are nonpermissive for ecotropic SL3. A
second means was to introduce a frameshift mutation by filling in the
cohesive ends of a unique XhoI site in the middle of the
RT-encoding portion of the pol gene of the full-length viral
clones that contained the intact and mutated R regions (Fig. 1A). The
latter approach was used for the experiment in mouse cells. The
constructs were transfected into human K562 cells, as previously
described (15, 16). This line was chosen because earlier
work showed that the RSL had a large effect here (15, 16).
Dishes (100-mm diameter) containing 106 cells were
transfected with 10 µg of plasmid DNA by the DEAE-dextran method as
previously described (15, 16). Cells from three plates were
pooled and collected 2 days after transfection. Duplicates were
performed with parallel sets of three plates each. The cells were lysed
with 0.5% Nonidet P-40. Nuclei and microsomes were pelleted by
centrifugation at 13,000 × g for 10 min. Supernatants were phenol-chloroform extracted, and RNA was collected by ethanol precipitation. Cytoplasmic RNA (10 µg) were electrophoresed in 1%
agarose gels. Blotting and hybridization were performed as described
previously (47). The viral probe was a 630-bp
BglII fragment from the SU portion of the env
gene of SL3. The blots were rehybridized to a mouse
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
R Region Sequences in the Long Terminal Repeat of a
Murine Retrovirus Specifically Increase Expression of
Unspliced RNAs
and
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
G of this secondary structure
was 
7.5 kcal/mol. Phylogenetic analysis indicated that the sequences
important for the maintenance of this secondary structure were
conserved among mammalian type C retroviruses. Mutations that disrupted
secondary structure decreased the level of expression of a reporter
gene under the control of viral LTR sequences about 10-fold in
transient expression assays and 20-fold in fibroblasts stably
transformed with the LTR reporter plasmids (16). A
compensatory mutant in which the nucleotide sequence of the stem was
different but the predicted secondary structure was maintained had most
of the activity of the wild-type LTR (16). Thus, the
stem-loop structure was important for the maximum activity of the SL3
LTR and presumably for the LTRs of other type C mammalian retroviruses.
For simplicity, we term the R region stem-loop element the RSL.
-actin probe
(Ambion) to control for the relative amounts of RNA loaded in the
different lanes. Hybridization was visualized and quantified by
PhosphorImager analysis. Two bands were visible on the Northern blot,
corresponding to the full-length transcript and the spliced
env mRNA. The results observed in K562 cells are shown in
Fig. 1B. Similar results were observed in NIH 3T3 cells (8,
68).
View larger version (47K):
[in a new window]
FIG. 1.
(A) Plasmid clone of a full-length SL3 genome. The clone
contains a wild-type 3' LTR and either a wild-type 5' LTR or the
RSL-deleted 5' LTR. For experiments in mouse cells that were permissive
for viral replication, a frameshift mutation was introduced into the
unique XhoI site in the portion of pol that
encodes RT. (B) Northern blot analysis of transfected K562 cells with
viral RNAs transcribed from the full-length viral templates that
contained either a wild-type 5' LTR or the RSL-deleted 5' LTR. Control,
mock-transfected cells. SL3, an intact 5' LTR. S R, a 5' LTR with a
deletion of the RSL. 1 and 2, duplicate transfection of the SL3 or
SR templates. The slower migrating bands are the unspliced
full-length transcripts. The faster migrating bands are the spliced
env mRNAs. R shows the position of the RSL deletion
relative to wild-type (WT) SL3. Stop indicates a stop codon due to the
frameshift mutation introduced at the XhoI site.
View larger version (33K):
[in a new window]
FIG. 2.
(A) Plasmids used to test the effects of the RSL on
spliced and unspliced transcripts. The top diagram shows the
pCAT3-Basic plasmid with the SL3 LTR inserted upstream of the intron.
SD, splice donor; SA, splice acceptor. The second diagram shows the
same plasmid after the intron was deleted by excision of a
HindIII fragment. The bottom part of the figure shows
the sequences of the first 32 nucleotides of the R region of SL3 and
the four mutants that were tested. Names at the left of each line
identify the mutations: S R, SL3 RSL deletion; ASC, ascending side of
the stem; DES, descending side of the stem; COMP, compensatory. The
nucleotides that comprise the stem and the loop are indicated. The
large dots above the SL3 sequence indicate the nucleotides that are
involved in base pairing (15, 16). The underlined, small
dots in the SR sequence indicate the nucleotides that were deleted.
The underlined nucleotides in the ASC, DES, and COMP mutants indicate
the positions of the substitutions. The symbols to the right of each
sequence represent the structures into which the nucleotides are
predicted to fold at the 5' ends of the transcribed RNAs. Stars
indicate the positions of the substitutions of five consecutive
nucleotides. (B) Structures of the SL3 CAT plasmids used in earlier
studies (15, 16). The top diagram shows the structure of a
plasmid that contained U3, R, U5, and 258 nucleotides of 5'
untranslated (5'UT) sequences downstream of the LTR. The bottom diagram
shows the structure of a plasmid that contained U3 and R sequences.
These plasmids were derived from the CAT plasmids of Gorman et al.
(30).
-actin probe, the
level of the full-length transcript from virus with the RSL deletion
was over fivefold lower than that from wild-type SL3. The levels of the
spliced envelope transcript were similar in cells transfected with the
wild-type construct and in those transfected with RSL-deleted viral
constructs. These results showed that the SL3 RSL did function in the
context of the full-length viral genome. However, it only affected the
cytoplasmic levels of the unspliced full-length transcript.
Use of a CAT reporter plasmid system to test the effect of mutations in the SL3 RSL on spliced versus unspliced transcripts. We wished to confirm the observation that the SL3 RSL specifically affected the cytoplasmic levels of unspliced mRNAs by using a second experimental approach. Therefore, a reporter gene system was devised to assess the effect of the R region on transcripts that differed by the presence or absence of an intron. This system provided a simple way to compare the effects of a set of key mutations within the RSL on spliced and unspliced RNA. Specifically, we wanted to test whether mutations that disrupted the secondary structure of the RSL specifically affected unspliced transcripts. Chloramphenicol acetyltransferase (CAT) reporter constructs were engineered that were isogenic except for the presence of an intron (Fig. 2A). Transcripts containing the intron should be spliced, whereas transcripts from the template with the deleted intron should not be spliced.
The pCAT3-Basic vector was utilized for this purpose. This plasmid contains a cloning site for promoter sequences and the CAT gene (Fig. 2A). The viral LTR was inserted at the cloning site (Fig. 2A). Between the LTR and the CAT gene, the plasmid contains an intron (Fig. 2A). The intron was composed of the donor site from the first intron of the human-globin gene and the branch and acceptor sites from the intron
of an immunoglobulin gene. The sequences of the donor, acceptor, and
branchpoint sites were optimized to match the consensus sequences
(9, 63). The intron is flanked by HindIII
sites (Fig. 3) and thus was easily removed to generate intronless plasmids. The vector also contained a
simian virus 40 (SV40) late polyadenylation sequence 3' to the CAT
reporter gene (Fig. 2A).
|
R, was
the same deletion of 28 nucleotides (positions +4 to +31, inclusive) of
the SL3 RSL that was tested in the full-length viral constructs. To
test whether the secondary structure of the RSL was important for the
effect on the spliced cytoplasmic RNA, mutations that disrupted the
stem-loop structure were also tested. These were a mutation of five
nucleotides in the upper portion of the ascending side of the stem
(ASC) and a mutation in the corresponding five nucleotides in the
descending side of the stem (DES). These mutations disrupted most of
the base pairs of the original stem-loop structure. The last mutation (COMP) included both the ASC and the DES mutations. These were compensatory mutations that altered the nucleotide sequence of the stem
but restored the predicted secondary structure.
Effects of the mutations in the SL3 RSL on spliced and unspliced transcripts. To test if the mutations of the SL3 RSL had different effects on spliced versus unspliced transcripts, the CAT reporter constructs, with or without the intron, were transiently transfected into cells and CAT activity was determined. Three different cell lines were tested. The first was NIH 3T3, a fibroblast cell line where the original effect of RSL mutations was observed. This line was chosen because it was previously observed that the effect of the RSL mutations was greater in fibroblasts than in any other type of cell tested (15). Since SL3 virus causes T-cell lymphoma, two T-cell lymphoma lines, L691 and Jurkat, were also tested. NIH 3T3 cells were transfected with Lipofectamine Plus (Gibco-BRL). L691 and Jurkat cells were transfected with DEAE-dextran (15, 16). Transfections were performed with 5 µg of LTR-CAT plasmid DNA plus 1 µg of Rous sarcoma virus LTR-luciferase plasmid as a control for transfection efficiency. Each transfection was performed in duplicate on at least two separate occasions. BODIPY FL chloramphenicol (Molecular Probes)-labeled products were resolved by thin-layer chromatography and then visualized and quantified by PhosphorImager (Molecular Dynamics) analysis. CAT activity for each sample was normalized to the luciferase activity of a cotransfected control plasmid to correct for variations in transfection efficiency. Then the means were calculated for the multiple trials of a particular plasmid in each specific cell line. Activities were expressed relative to the wild-type LTR.
When the constructs containing an intron were tested, the RSL was found to have relatively small effects in all three cell lines (Fig. 3A). The LTR with the deletion of the RSL exhibited 44 to 63% of the activity of the wild-type LTR. Mutations in either the ascending or the descending side of the stem resulted in activity similar to that of the LTR with a deletion of the RSL, although in the NIH 3T3 cells, the LTR with the descending stem mutation was only about half as active as SR. The LTR with the compensatory mutation in the RSL exhibited
activity that ranged between those exhibited by the wild-type and
RSL-deleted LTRs. Thus, the RSL had little effect on reporter
gene-containing transcripts that also contained an efficiently excised intron.
In contrast, when the intron was not present, the effects of the RSL
were quite pronounced (Fig. 3B). Transcripts that did not contain the
intron were expected not to be spliced. When the intron was deleted in
the construct with the wild-type SL3 LTR, activity was reduced about
30-fold compared to the activity of the intron-containing construct in
the different cell lines (Fig. 3). This was consistent with the idea
that splicing is generally important for the efficient expression of
genes (10-12, 33, 34, 43).
When mutations of the RSL were tested in the intronless template, large
effects were observed (Fig. 3B). In NIH 3T3 cells, the LTR with the
deletion of the RSL was 60-fold less active than the wild-type LTR. In
the other cell lines, the effect was smaller but still very
significant. In L691 and Jurkat cells, the LTR with the deletion of the
RSL had 15 and 9% as much activity, respectively, as the LTR with an
intact RSL. Thus, the RSL had a large effect on the expression of the
reporter gene from the unspliced transcript.
We tested whether the effects of the RSL on the intronless transcripts
were dependent on the secondary structure of the RSL element. In all
cell lines tested, the LTRs with the ASC and DES mutations had effects
comparable to the SR LTR (Fig. 3B). In NIH 3T3 cells, the activity
of the LTR with the disrupted secondary structure was reduced to a
level comparable to that of the pCAT3-Basic vector with no promoter
driving expression of the CAT gene. This indicated that the RSL was
essential for expression of the reporter gene in the unspliced
transcript in this cell line.
Restoration of the secondary structure with the COMP mutation restored
activity to 45 to 60% of the level of the wild-type LTR in all three
cell lines (Fig. 3B). Thus, the secondary structure of the RSL was
important for the effect of the RSL on the unspliced transcript.
In summary, the RSL had little effect on spliced transcripts. When
splicing was prevented by removal of the intron, the level of
expression of the reporter gene was greatly reduced. Thus, expression
of the reporter gene in unspliced transcripts was highly dependent on
the RSL. In particular, it required the secondary structure formed by
the RSL at the 5' end of the transcript.
Implications for the mechanisms of action of the RSL. Our experiments provided two lines of evidence that the RSL specifically affected unspliced transcripts. The Northern blot analysis of transcripts of the SL3 genome showed that the RSL was required for cytoplasmic accumulation of the unspliced full-length transcript. The levels of the spliced transcript remained unchanged. The specificity of the RSL for unspliced transcripts was demonstrated further by analyzing its effects on transcripts that were engineered to be either efficiently spliced or not spliced. The RSL had large effects only on the unspliced transcripts from an intronless template. Little to no effect was seen on the efficiently spliced transcripts. Therefore, we conclude that the SL3 RSL plays a role in the cytoplasmic accumulation of unspliced viral transcripts.
An obligatory aspect of the life cycles of all retroviruses is that a fraction of the viral full-length transcripts must be transported to the cytoplasm without being spliced. The unspliced RNA functions both as the mRNA for the gag, pro, and pol genes and as the genome that is packaged into viral particles. In simple retroviruses, a single splice donor is positioned near the 5' end of the primary transcript and splicing involves joining to acceptor sites positioned downstream in the RNA. One mechanism that results in only a fraction of the primary transcripts being spliced is that the splicing signals of retroviruses are suboptimal (11, 27, 39, 72). Additional sequences in the genomes of simple retroviruses besides the donor, acceptor, and branchpoint elements also affect the levels of spliced and unspliced transcripts (2, 3, 29, 48, 49, 52, 54). Our Northern blot analysis of transcripts from the full-length viral genome showed that the SL3 RSL can increase the fraction of cytoplasmic, viral RNA that is unspliced. Since the equivalent sequences from other type C mammalian retroviruses can substitute for the SL3 RSL and form similar stem-loop structures (15, 16), we hypothesize that the effect of the RSL on unspliced RNA may be a general feature of this genus of retroviruses. Complex retroviruses control the cytoplasmic accumulation of unspliced and partially spliced RNAs by encoding regulatory proteins such as Rev for HIV-1 and Rex for human T-cell leukemia virus (32, 33, 46). These proteins interact with cis-acting elements in the transcripts and mediate the export of the partially spliced and unspliced RNAs from the nucleus. Due to the presence of suboptimal splicing signals, cellular splicing factors are thought to bind to inefficiently spliced RNAs and prevent them from leaving the nucleus (11, 39, 43). Binding of HIV-1 Rev promotes the transport of the unspliced RNAs out of the nucleus (6, 11, 18, 23-26, 45, 46, 65, 66, 71). In contrast, transcripts containing optimal splicing signals are efficiently exported to the cytoplasm (11, 43). These observations are consistent with the idea that splicing and cytoplasmic transport may be coupled. Simple retroviruses do not encode proteins that promote nuclear export. However, some have been shown to contain cis regulatory elements that allow the transport of unspliced RNAs to the cytoplasm. Well-studied examples of such elements exist between the env gene and 3' LTR in Mason-Pfizer monkey virus and simian retrovirus type 1 (7, 22, 33, 67, 74). This element, known as the constitutive transport element, is capable of substituting for the REV response element. Elements with similar functions were identified in hepatitis B virus, woodchuck hepatitis virus, avian leukosis virus, and Rous sarcoma virus (20, 21, 35, 51, 52). Presumably, cellular factors recognize these elements in the viral RNA and mediate nuclear export of the RNA (55). There are three possible mechanisms by which the RSL of MuLVs might function to increase the cytoplasmic levels of unspliced transcripts. It could play a direct role in the transport of the unspliced transcripts to the cytoplasm in a manner analogous to the constitutive transport element of Mason-Pfizer monkey virus and the RSV element. If the SL3 RSL is indeed a cytoplasmic transport element, then it is located at a different position in the viral genome than the elements identified in other viruses. These are generally located in the 3' portion of the viral transcripts. It remains to be investigated whether SL3 also contains a 3' element that affects cytoplasmic levels of unspliced RNAs. A second possible model is that the secondary structure of the SL3 RSL may interfere with a 5' exoribonuclease. Stabilization against degradation in the nucleus would then allow the transcripts more chances to be exported to the cytoplasm. There are 5' exoribonucleases known to exist in Saccharomyces cerevisiae (57, 61). For such an enzyme to account for the effects observed here, it must preferentially degrade unspliced transcripts. This might occur if the unspliced transcripts are detained in the nucleus and the exoribonuclease is specifically localized in the nucleus. The stem-loop structure at the 5' end of SL3 LTR-driven transcripts might play a role in blocking the activity of such a 5' exoribonuclease. However, we postulate that it is unlikely that the SL3 RSL functions simply as an RNA structure at the 5' end of viral transcripts that blocks the progression of a 5' exoribonuclease. The approximately 600-nucleotide 5' untranslated sequence of MuLVs is predicted to fold into additional stem-loop structures (1) that the exoribonuclease would also have to overcome. The SL3 RSL has a predicted stability of only7.5
kcal/mol (15). More stable stem-loop structures could not functionally substitute for the SL3 RSL (17). We hypothesize that it is more likely that the stem-loop structure at the 5' end of
SL3 transcripts is recognized by a cellular factor that acts to block
the activity of a 5' exoribonuclease and/or promote nuclear export of
viral RNA.
A third possible mechanism by which the RSL might function is by
somehow impeding the splicing process. Perhaps a cellular factor
recognizes the RSL and by some mechanism directly interferes with the
activity of the cellular splicing apparatus.
In our initial reports, deletion of the SL3 RSL resulted in about a
10-fold decline in the expression of a CAT reporter gene in transient
expression assays in NIH 3T3 fibroblasts (15, 16). In the
same cells, deletion of the RSL resulted in a 60-fold decline in
expression of the CAT reporter gene transcribed from the intronless construct (Fig. 3B). The RSL had only a twofold effect on the reporter
gene transcribed from the template that contained an efficiently
spliced intron (Fig. 3A). Since the latter two templates were isogenic
except for the intron, the intron must account for the difference in
activity of the RSL in the two constructs. The intermediate level of
activity of the RSL in the constructs used in our earlier studies
(15, 16) might also be due to the effects of splicing
signals. These constructs contained the SV40 small-t antigen splice
donor and acceptor (Fig. 2B). SV40 early RNA is spliced with one of two
alternative donors and a common acceptor (28, 73). We
hypothesize that this transcript is spliced with an efficiency that is
intermediate between those of the efficiently spliced transcripts from
the intron-containing pCAT3-Basic plasmid and the unspliced transcripts
from the intronless plasmid. The RSL would then function on these
transcripts to increase their cytoplasmic levels. Similarly, the
fivefold effect of the SL3 RSL on the full-length viral transcript
presumably reflected the suboptimal splicing signals known to be
present in retroviral transcripts.
Whatever the mechanism by which the SL3 RSL functions, it is clear that
the secondary structure of the stem is crucial for the effect.
Mutations that disrupted base pairing of the RSL reduced the expression
of the reporter gene from the intronless template to a level comparable
to that for the deletion of the entire RSL (Fig. 3B). Restoration of
base pairing by the compensatory mutation restored most of the activity
of the element. We hypothesize that this stem structure is recognized
by a cellular factor that mediates the nuclear export of transcripts,
protects the transcripts from a nuclear 5' exoribonuclease, and/or
interferes with splicing.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by NIH grants CA44822 and CA57337 to J.L. A.M.T. was supported by NIH training grant GM7288. Core facilities for oligonucleotide synthesis, PhosphorImager analysis, and DNA sequencing were supported by NIH Cancer Center grant CA13330 and NIH Center for AIDS Research grant AI27741 to the Albert Einstein College of Medicine.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3715. Fax: (718) 430-8778. E-mail: lenz{at}aecom.yu.edu.
Present address: Regeneron Pharmaceuticals, Tarrytown, NY 10591.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Aiyar, A.,
D. Cobrinik,
Z. Ge,
H.-J. Kung, and J. Leis.
1992.
Interaction between retroviral U5 RNA and the T C loop of the tRNATrp primer is required for efficient initiation of reverse transcription.
J. Virol.
66:2464-2472 |
| 2. | Amendt, B. A., S. B. Simpson, and C. M. Stoltzfus. 1995. Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor. J. Virol. 69:5068-5076[Abstract]. |
| 3. |
Arrigo, S., and K. L. Beemon.
1988.
Regulation of Rous sarcoma virus RNA splicing and stability.
Mol. Cell. Biol.
8:4858-4867 |
| 4. |
Berkhout, B.
1992.
Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.
Nucleic Acids Res.
20:27-31 |
| 5. | Berkhout, B., R. H. Silverman, and K. Jeang. 1989. Tat transactivates the human immunodeficiency virus through a nascent RNA target. Cell 59:273-282[Medline]. |
| 6. | Bogerd, H. P., R. A. Fridell, S. Madore, and B. R. Cullen. 1995. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 82:485-494[Medline]. |
| 7. |
Bray, M.,
S. Prasad,
J. W. Dubay,
E. Hunter,
K. T. Jeang,
D. Rekosh, and M. L. Hammarskjold.
1994.
A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent.
Proc. Natl. Acad. Sci. USA
91:1256-1260 |
| 8. |
Brinster, R. L.,
J. D. Allen,
R. R. Behringer,
R. E. Gelinas, and R. D. Palmiter.
1988.
Introns increase transcriptional efficiency in transgenic mice.
Proc. Natl. Acad. Sci. USA
85:836-840 |
| 9. | Brondyk, B. 1994. pCI and pSI mammalian expression vectors. Promega Notes 49:7-11. |
| 10. |
Buchman, A. R., and P. Berg.
1988.
Comparison of intron-dependent and intron-independent gene expression.
Mol. Cell. Biol.
8:4395-4405 |
| 11. | Chang, D. D., and P. A. Sharp. 1989. Regulation by HIV Rev depends upon recognition of splice sites. Cell 59:789-795[Medline]. |
| 12. |
Choi, T.,
M. Huang,
C. Gorman, and R. Jaenisch.
1991.
A generic intron increases expression in transgenic mice.
Mol. Cell. Biol.
11:3070-3074 |
| 13. |
Cujec, T. P.,
H. Okamoto,
K. Fujinaga,
J. Meyer,
H. Chamberlin,
D. O. Morgan, and B. M. Peterlin.
1997.
The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II.
Genes Dev.
11:2645-2657 |
| 14. | Cullen, B. R. 1990. The HIV-1 tat protein: an RNA sequence-specific processivity factor? Cell 63:655-657[Medline]. |
| 15. |
Cupelli, L., and J. Lenz.
1991.
Transcriptional initiation and postinitiation effects of murine leukemia virus long terminal repeat R-region sequences.
J. Virol.
65:6961-6968 |
| 16. |
Cupelli, L.,
S. A. Okenquist,
A. Trubetskoy, and J. Lenz.
1998.
The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression.
J. Virol.
72:7807-7814 |
| 17. | Cupelli, L. A., and J. Lenz. Unpublished results. |
| 18. | Daly, T. J., K. S. Cook, G. S. Gray, T. E. Maione, and J. R. Rusche. 1989. Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro. Nature 342:816-819[Medline]. |
| 19. |
Derse, D., and J. Casey.
1986.
Two elements in the bovine leukemia virus long terminal repeat that regulate gene expression.
Science
231:1437-1440 |
| 20. | Donello, J. E., A. A. Beeche, G. J. Smith, G. R. Lucero, and T. J. Hope. 1996. The hepatitis B virus posttranscriptional regulatory element is composed of two subelements. J. Virol. 70:4345-4351[Abstract]. |
| 21. |
Donello, J. E.,
J. E. Loeb, and T. J. Hope.
1998.
Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element.
J. Virol.
72:5085-5092 |
| 22. | Ernst, R. K., M. Bray, D. Rekosh, and M. L. Hammarskjold. 1997. A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol. Cell. Biol. 17:135-144[Abstract]. |
| 23. | Fischer, U., J. Huber, W. C. Boelens, I. W. Mattaj, and R. Luhrmann. 1995. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475-483[Medline]. |
| 24. |
Fridell, R. A.,
H. P. Bogerd, and B. R. Cullen.
1996.
Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.
Proc. Natl. Acad. Sci. USA
93:4421-4424 |
| 25. | Fritz, C. C., and M. R. Green. 1996. HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs. Curr. Biol. 6:848-854[Medline]. |
| 26. | Fritz, C. C., M. L. Zapp, and M. R. Green. 1995. A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 376:530-533[Medline]. |
| 27. |
Fu, X. D.,
R. A. Katz,
A. M. Skalka, and T. Maniatis.
1991.
The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.
Genes Dev.
5:211-220 |
| 28. |
Fu, X. Y.,
J. D. Colgan, and J. L. Manley.
1988.
Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.
Mol. Cell. Biol.
8:3582-3590 |
| 29. |
Gontarek, R. R.,
M. T. McNally, and K. L. Beemon.
1993.
Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing.
Genes Dev.
7:1926-1936 |
| 30. |
Gorman, C. M.,
L. F. Moffat, and B. H. Howard.
1982.
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol. Cell. Biol.
2:1044-1051 |
| 31. |
Hauber, J., and B. R. Cullen.
1988.
Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type 1 long terminal repeat.
J. Virol.
62:673-679 |
| 32. | Hidaka, M., J. Inoue, M. Yoshida, and M. Seiki. 1988. Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. EMBO J. 7:519-523[Medline]. |
| 33. | Hope, T. J. 1997. Viral RNA export. Chem. Biol. 4:335-344[Medline]. |
| 34. |
Huang, M., and C. Gorman.
1990.
Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.
Nucleic Acids Res.
18:937-947 |
| 35. |
Huang, Z. M., and T. S. Yen.
1994.
Hepatitis B virus RNA element that facilitates accumulation of surface gene transcripts in the cytoplasm.
J. Virol.
68:3193-3199 |
| 36. |
Inoue, J.,
M. Yoshida, and M. Seiki.
1987.
Transcriptional (p40x) and posttranscriptional (p27x-III) regulators are required for the expression and replication of human T-cell leukemia virus type I genes.
Proc. Natl. Acad. Sci. USA
84:3653-3657 |
| 37. |
Jones, K. A.,
P. A. Luciw, and N. Duchange.
1988.
Structural arrangements of transcription control domains within the 5'-untranslated leader regions of the HIV-1 and HIV-2 promoters.
Genes Dev.
2:1101-1114 |
| 38. |
Kato, H.,
H. Sumimoto,
P. Pognonec,
C. H. Chen,
C. A. Rosen, and R. G. Roeder.
1992.
HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.
Genes Dev.
6:655-666 |
| 39. |
Katz, R. A., and A. M. Skalka.
1990.
Control of retroviral RNA splicing through maintenance of suboptimal processing signals.
Mol. Cell. Biol.
10:696-704 |
| 40. | Kiss-Toth, E., and I. Unk. 1994. A downstream regulatory element activates the bovine leukemia virus promoter. Biochem. Biophys. Res. Commun. 202:1553-1561[Medline]. |
| 41. | Laspia, M. F., A. P. Rice, and M. B. Mathews. 1989. HIV-1 tat protein increases transcriptional initiation and stabilizes elongation. Cell 59:283-292[Medline]. |
| 42. |
Laspia, M. F.,
A. P. Rice, and M. B. Mathews.
1990.
Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation.
Genes Dev
4:2397-2408 |
| 43. | Legrain, P., and M. Rosbash. 1989. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57:573-583[Medline]. |
| 44. |
Lobel, L. I., and S. P. Goff.
1985.
Reverse transcription of retroviral genomes: mutations in the terminal repeat sequences.
J. Virol.
53:447-455 |
| 45. |
Malim, M. H., and B. R. Cullen.
1993.
Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.
Mol. Cell. Biol.
13:6180-6189 |
| 46. | Malim, M. H., J. Hauber, S. Le, J. Maizel, and B. R. Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338:254-257[Medline]. |
| 47. | Marciniak, R. A., and P. A. Sharp. 1991. HIV-1 Tat protein promotes formation of more-processive elongation complexes. EMBO J. 10:4189-4196[Medline]. |
| 48. |
McNally, M. T., and K. L. Beemon.
1992.
Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.
J. Virol.
66:6-11 |
| 49. |
Miller, J. T., and C. M. Stoltzfus.
1992.
Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA.
J. Virol.
66:4242-4251 |
| 50. | Montagne, J., and P. Jalinot. 1995. Characterization of a transcriptional attenuator within the 5' R region of the human T cell leukemia virus type 1. AIDS Res. Hum. Retroviruses 11:1123-1129[Medline]. |
| 51. |
Ogert, R. A., and K. L. Beemon.
1998.
Mutational analysis of the Rous sarcoma virus DR posttranscriptional control element.
J. Virol.
72:3407-3411 |
| 52. | Ogert, R. A., L. H. Lee, and K. L. Beemon. 1996. Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm. J. Virol. 70:3834-3843[Abstract]. |
| 53. | Ohtani, K., M. Nakamura, S. Saito, T. Noda, Y. Ito, K. Sugamura, and Y. Hinuma. 1987. Identification of two distinct elements in the long terminal repeat of HTLV-I responsible for maximum gene expression. EMBO J. 6:389-395[Medline]. |
| 54. |
Oshima, M.,
T. Odawara,
K. Hanaki,
H. Igarashi, and H. Yoshikura.
1998.
cis elements required for high-level expression of unspliced gag-containing message in Moloney murine leukemia virus.
J. Virol.
72:6414-6420 |
| 55. | Pasquinelli, A. E., R. K. Ernst, E. Lund, C. Grimm, M. L. Zapp, D. Rekosh, M. L. Hammarskjold, and J. E. Dahlberg. 1997. The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16:7500-7510[Medline]. |
| 56. |
Pierce, J.,
B. E. Fee,
M. G. Toohey, and D. O. Peterson.
1993.
A mouse mammary tumor virus promoter element near the transcription initiation site.
J. Virol.
67:415-424 |
| 57. | Poole, T. L., and A. Stevens. 1997. Structural modifications of RNA influence the 5' exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 235:799-805[Medline]. |
| 58. |
Ridgway, A. A. G.,
H.-J. Kung, and D. J. Fujita.
1989.
Transient expression analysis of the reticuloendotheliosis virus long terminal repeat element.
Nucleic Acids Res.
17:3199-3215 |
| 59. | Rosen, C. A., J. Sodroski, and W. A. Haseltine. 1985. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell 41:813-823[Medline]. |
| 60. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 61. |
Schwer, B.,
X. Mao, and S. Shuman.
1998.
Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme.
Nucleic Acids Res.
26:2050-2057 |
| 62. | Selby, M. J., and B. M. Peterlin. 1990. Trans-activation by HIV-1 Tat via a heterologous RNA binding protein. Cell 62:769-776[Medline]. |
| 63. | Senapathy, P., M. B. Shapiro, and N. L. Harris. 1990. Splice junctions, branch point sites, and exons: sequence statistics, identification, and application to genome project. Methods Enzymol. 183:252-278[Medline]. |
| 64. | Southgate, C., M. L. Zapp, and M. R. Green. 1990. Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein. Nature 345:640-642[Medline]. |
| 65. | Stutz, F., E. Izaurralde, I. W. Mattaj, and M. Rosbash. 1996. A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus. Mol. Cell. Biol. 16:7144-7150[Abstract]. |
| 66. | Stutz, F., M. Neville, and M. Rosbash. 1995. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell 82:495-506[Medline]. |
| 67. | Tabernero, C., A. S. Zolotukhin, A. Valentin, G. N. Pavlakis, and B. K. Felber. 1996. The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J. Virol. 70:5998-6011[Abstract]. |
| 68. | Trubetskoy, A., S. Okenquist, and J. Lenz. Unpublished results. |
| 69. | Wei, P., M. E. Garber, S. M. Fang, W. H. Fischer, and K. A. Jones. 1998. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451-462[Medline]. |
| 70. |
Yang, X.,
M. O. Gold,
D. N. Tang,
D. E. Lewis,
E. Aguilar-Cordova,
A. P. Rice, and C. H. Herrmann.
1997.
TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinase and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines.
Proc. Natl. Acad. Sci. USA
94:12331-12336 |
| 71. | Zapp, M. L., and M. R. Green. 1989. Sequence-specific RNA binding by the HIV-1 Rev protein. Nature 342:816-819. |
| 72. | Zhang, L., and C. M. Stoltzfus. 1998. A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus. Virology 206:1099-1107. |
| 73. |
Zhuang, Y.,
H. Leung, and A. M. Weiner.
1987.
The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA.
Mol. Cell. Biol.
7:3018-3020 |
| 74. |
Zolotukhin, A. S.,
A. Valentin,
G. N. Pavlakis, and B. K. Felber.
1994.
Continuous propagation of RRE() and Rev()RRE() human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.
J. Virol.
68:7944-7952 |
Journal of Virology, April 1999, p. 3477-3483, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Groom, H. C. T., Anderson, E. C., Lever, A. M. L.
(2009). Rev: beyond nuclear export. J. Gen. Virol.
90: 1303-1318
[Abstract] [Full Text] -
Kraunus, J., Zychlinski, D., Heise, T., Galla, M., Bohne, J., Baum, C.
(2006). Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5{middle dot} Splice Site. J. Biol. Chem.
281: 37381-37390
[Abstract] [Full Text] -
Metzl, C., Mischek, D., Salmons, B., Gunzburg, W. H., Renner, M., Portsmouth, D.
(2006). Tissue- and Tumor-Specific Targeting of Murine Leukemia Virus-Based Replication-Competent Retroviral Vectors. J. Virol.
80: 7070-7078
[Abstract] [Full Text] -
Swindle, C. S., Kim, H. G., Klug, C. A.
(2004). Mutation of CpGs in the Murine Stem Cell Virus Retroviral Vector Long Terminal Repeat Represses Silencing in Embryonic Stem Cells. J. Biol. Chem.
279: 34-41
[Abstract] [Full Text] -
Heath, M. J., Derebail, S. S., Gorelick, R. J., DeStefano, J. J.
(2003). Differing Roles of the N- and C-terminal Zinc Fingers in Human Immunodeficiency Virus Nucleocapsid Protein-enhanced Nucleic Acid Annealing. J. Biol. Chem.
278: 30755-30763
[Abstract] [Full Text] -
Wilson, C. A., Laeeq, S., Ritzhaupt, A., Colon-Moran, W., Yoshimura, F. K.
(2002). Sequence Analysis of Porcine Endogenous Retrovirus Long Terminal Repeats and Identification of Transcriptional Regulatory Regions. J. Virol.
77: 142-149
[Abstract] [Full Text] -
Scheef, G., Fischer, N., Flory, E., Schmitt, I., Tonjes, R. R.
(2002). Transcriptional Regulation of Porcine Endogenous Retroviruses Released from Porcine and Infected Human Cells by Heterotrimeric Protein Complex NF-Y and Impact of Immunosuppressive Drugs. J. Virol.
76: 12553-12563
[Abstract] [Full Text] -
Hull, S., Boris-Lawrie, K.
(2002). RU5 of Mason-Pfizer Monkey Virus 5' Long Terminal Repeat Enhances Cytoplasmic Expression of Human Immunodeficiency Virus Type 1 gag-pol and Nonviral Reporter RNA. J. Virol.
76: 10211-10218
[Abstract] [Full Text] -
Williams, M. C., Gorelick, R. J., Musier-Forsyth, K.
(2002). Specific zinc-finger architecture required for HIV-1 nucleocapsid protein's nucleic acid chaperone function. Proc. Natl. Acad. Sci. USA
99: 8614-8619
[Abstract] [Full Text] -
Furger, A., Monks, J., Proudfoot, N. J.
(2001). The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation. J. Virol.
75: 11735-11746
[Abstract] [Full Text] -
Russell, R. A., Zeng, Y., Erlwein, O., Cullen, B. R., McClure, M. O.
(2001). The R Region Found in the Human Foamy Virus Long Terminal Repeat Is Critical for both Gag and Pol Protein Expression. J. Virol.
75: 6817-6824
[Abstract] [Full Text] -
Dang, Q., Hu, W.-S.
(2001). Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication. J. Virol.
75: 809-820
[Abstract] [Full Text] -
Roberts, T. M., Boris-Lawrie, K.
(2000). The 5' RNA Terminus of Spleen Necrosis Virus Stimulates Translation of Nonviral mRNA. J. Virol.
74: 8111-8118
[Abstract] [Full Text] -
Heinkelein, M., Thurow, J., Dressler, M., Imrich, H., Neumann-Haefelin, D., McClure, M. O., Rethwilm, A.
(2000). Complex Effects of Deletions in the 5' Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging. J. Virol.
74: 3141-3148
[Abstract] [Full Text] -
Martiney, M. J., Rulli, K., Beaty, R., Levy, L. S., Lenz, J.
(1999). Selection of Reversions and Suppressors of a Mutation in the CBF Binding Site of a Lymphomagenic Retrovirus. J. Virol.
73: 7599-7606
[Abstract] [Full Text] -
Zhou, T., Chiang, CM
(2001). The Intronless and TATA-less Human TAFII55 Gene Contains a Functional Initiator and a Downstream Promoter Element. J. Biol. Chem.
276: 25503-25511
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»