JOURNAL OF VIROLOGY, Apr. 2002, p. 3292–3300 Vol. 76, No. 7
0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.7.3292–3300.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Nuclear Interactions Are Necessary for Translational Enhancement
by Spleen Necrosis Virus RU5
Andrew W. Dangel,
Tifﬁney M. Roberts,
and Kathleen Boris-Lawrie
Center for Retrovirus Research,
Departments of Veterinary Biosciences
and Molecular Virology, Immunology
and Medical Genetics,
Molecular, Cellular and Developmental Biology Graduate Program,
Comprehensive Cancer Center,
The Ohio State University, Columbus, Ohio 43210-1093
Received 17 September 2001/Accepted 4 January 2002
The 5ⴕ long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-
independent expression of intron-containing human immunodeﬁciency virus type 1 (HIV-1) gag. The SNV RU5
region, which corresponds to the 165-nucleotide 5ⴕ RNA terminus, functions in a position- and orientation-
dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc
RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for
nuclear export, efﬁcient translation, and nonsense-mediated decay. This project addressed the relationship
between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of
RNA transfection experiments suggest that cytoplasmic proteins are insufﬁcient for SNV RU5 translational
enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase
protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the
LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1
RRE. As a unique tool with which to investigate the relationship between different RNA trafﬁcking routes and
translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a
less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the
CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study
is the ﬁrst to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that
is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.
Retroviruses contain structured RNA elements that interact
with viral and cellular proteins and modulate nuclear export
and efﬁcient translation of intron-containing transcripts (re-
viewed in references 3, 5, and 11). The long terminal repeat
(LTR) of spleen necrosis virus (SNV) facilitates Rev/Rev-re-
sponsive element (RRE)-independent expression of unspliced
human immunodeﬁciency virus type 1 (HIV-1) gag reporter
RNA (7). Quantitative RNA and protein analysis identiﬁed a
minor 2-fold increase in cytoplasmic accumulation of gag RNA
but a greater-than-100-fold increase in Gag protein production
in response to the SNV LTR. The 165-nucleotide 5⬘ RNA
terminus encoded by the RU5 region of the LTR functions in
a position- and orientation-dependent manner to direct poly-
some association and detectable Gag protein synthesis. SNV
RU5 functions in a cap-dependent manner (M. Butsch and K.
Boris-Lawrie, unpublished data) and is not an internal ribo-
some entry site (IRES) (35). The SNV U5 region also aug-
ments translation of nonviral luciferase (luc) RNA by increas-
ing polysome loading (35). Recently, the R region of human
foamy virus (HFV) and RU5 of Mason-Pﬁzer monkey virus
(MPMV) were also shown to program unspliced gag RNA
templates for Gag protein synthesis (37; S. Hull and K. Boris-
Lawrie, submitted for publication). 5⬘-terminal translational
enhancers appear to be a mechanism shared among divergent
retroviruses to achieve efﬁcient expression of viral structural
protein. The relationship between nuclear export and produc-
tive cytoplasmic utilization directed by these 5⬘-terminal RNA
elements remains an important open question.
Evidence is mounting that nuclear association between nas-
cent RNAs and viral and cellular nuclear-cytoplasmic shuttling
proteins choreographs steps of posttranscriptional gene ex-
pression (reviewed in references 10, 28, and 38). The HIV-1
Rev protein is an essential activator of nuclear export and
cytoplasmic expression of intron-containing viral RNAs (14,
15, 20, 27). Rev interacts with newly synthesized RNA (23) by
binding to the RRE, which is within the terminal intron of
unspliced and singly spliced viral RNAs (9, 19, 21, 27, 36, 46).
Using a leucine-rich nuclear export sequence (NES), Rev acts
as an adapter protein that connects RRE-containing RNA to
the CRM1/exportin 1 nuclear export receptor (17, 30). CRM1
interacts with FG repeats of nucleoporins and exports RRE-
containing RNA by a leptomycin B (LMB)-inhibited pathway
that is typically reserved for 5S rRNA and cellular proteins that
contain leucine-rich NESs (16, 17). In the cytoplasm, Rev/
RRE augments RNA translational efﬁciency by directing poly-
some association (1, 7, 12, 26). Results of in situ hybridization
assays have shown that Rev transactivation correlates with
colocalization of RRE-containing RNAs with the cytoskeleton,
which is a supportive framework for interaction of polysomes
and mRNA (24, 25). It is not known whether nuclear export by
the CRM1-dependent pathway is necessary to program the
RRE-containing RNA for subcellular localization with cy-
toskeletal polysomes and efﬁcient translation.
This project addressed the relationship between the nuclear
export pathway and translational enhancement by SNV RU5.
* Corresponding author. Mailing address: Department of Veteri-
nary Biosciences, The Ohio State University, 1925 Coffey Rd., Colum-
bus, OH 43210-1093. Phone: (614) 292-1392. Fax: (614) 292-6473.
Results of RNA transfection and in vitro translation assays
indicate that cytoplasmic factors are insufﬁcient for SNV RU5
translational enhancement and suggest that nuclear interac-
tions are necessary. The nuclear export pathway of SNV RU5-
containing RNAs is not known. LMB sensitivity assays and
RNA analysis show that the nuclear export pathway of SNV
RU5 is distinct from the CRM1-dependent pathway accessed
by Rev/RRE. Upon combination of SNV RU5 and Rev/RRE
on a single gag RNA, we observe that Rev/RRE sequesters the
transcript to the leucine-rich CRM1-dependent nuclear export
pathway and that this nuclear export pathway abrogates trans-
lational enhancement by SNV RU5. Our results are consistent
with the model in which 5⬘-terminal SNV RU5 sequences and
nuclear factors direct access to a particular nuclear export
pathway that programs intron-containing RNA for efﬁcient
translation in the cytoplasm.
MATERIALS AND METHODS
Plasmids. The previously described plasmids used in this study are pYW99,
pYW208, pYW207, pYW205, pYW100, pGem (140-440), pMSBSVT7, pGAPDH
(7), pTR103, and pTR105 (35). Plasmids pYW100RRE, pYW205RRE, and
pTR155 were constructed from pYW100, pYW205, and pTR147, respectively, by
introduction of RRE into a SalI site in the 3⬘ untranslated region (UTR).
Plasmid pTR147 was constructed from pYW100 by exchange of the unique
BamHI/ApaI fragment from pYW207 that contains antisense RU5 and HIV 5⬘
UTR sequences. The RRE was ampliﬁed by PCR from HIV-1
7217 to 7689). pYW233 contains nine point mutations in RU5 that were intro-
duced into pYW100 by PCR-based site-directed mutagenesis (T. M. Roberts and
K. Boris-Lawrie, unpublished data). Plasmids pSVgagpolrre and pRev were
kindly provided by David Rekosh, University of Virginia (41).
In vitro transcriptions and RNase protection assays (RPAs). In the RNA
transfection and in vitro translation experiments, DNA templates for in vitro
transcription were prepared by PCR with primers that contain the T7 promoter
sequence incorporated at the 5⬘ terminus of the 5⬘ oligonucleotide. 5⬘-capped
transcripts were synthesized by T7 polymerase with mMessage mMachine (Am-
bion). The 3⬘ RNA terminus was polyadenylated in duplicate reactions with
poly(A) polymerase and either ATP or [␥-
P]ATP. The concentrations of the in
vitro transcripts were determined by spectrophotometry, and the quality was
veriﬁed by denaturing polyacrylamide gel electrophoresis of the
aliquot and PhosphorImager analysis with ImageQuant version 4.2 software
(Molecular Dynamics). RU5 gag and RU5 luc transcripts are 1,970 bases and
1,840 nucleotides in length, respectively.
For RPAs, antisense ␣-
P-labeled runoff transcripts were synthesized by
MAXIscript T7 RNA polymerase (Ambion) in accordance with the manufactur-
er’s instructions. A probe complementary to HIV
sequences in the 5⬘ UTR
common to each of the reporter RNAs was in vitro transcribed from template
pGEM(140-440) that had been linearized with NotI (7). A probe complementary
to cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was
transcribed from template pGAPDH, which had been linearized with NcoI. The
in vitro-transcribed RNAs were isolated by gel elution, and the RPAs were
performed with RPAIII (Ambion) with some modiﬁcations. Brieﬂy, 30 gof
cytoplasmic RNA or 15 g of nuclear RNA was precipitated with 3 ⫻ 10
of reporter probe and 2 ⫻ 10
cpm of the GAPDH probe. Samples were
hybridized at 42°C for 16 h. Samples were digested with an RNase mixture at
37°C for 30 min and then extracted with phenol-chloroform and chloroform and
precipitated with ethanol. Pellets were dissolved in loading buffer, heated at 90°C
for 3 min, and subjected to denaturing polyacrylamide gel electrophoresis on 5%
gels. RNase protection products were visualized by PhosphorImager analysis.
In vitro translations. RRLs (35 l; Promega) were programmed with up to
400 ng of capped and polyadenylated transcript in a total volume of 50 l and
incubated at 30°C for 1 h. Translation-competent 293 cell lysates were prepared
as described by Carroll and Lucas-Lenard without micrococcal nuclease treat-
ment (8) and were programmed with capped and polyadenylated transcript in a
total volume of 25 l and incubated at 30°C for up to 1 h. Programmed lysates
were directly assayed for Gag protein content or Luc activity.
Cells, transfections, and reporter assays. 293 cells are a human embryonic
kidney cell line. RNA transfections were performed on 293 cells with dilutions of
capped and polyadenylated transcript and 12 l of Lipofectin (Gibco-BRL) in 1
ml of serum-free Dulbecco modiﬁed Eagle medium. The luc and gag RNAs were
cotransfected and used reciprocally to normalize transfection efﬁciency. All
transfection assays were performed in triplicate and for at least three replicate
experiments. The Lipofectin-RNA mixtures were incubated with 293 cells (8 ⫻
) in 60-mm-diameter plates for 2 h and then replaced with Dulbecco modiﬁed
Eagle medium containing 10% fetal calf serum. Cells were harvested in phos-
phate-buffered saline (PBS) at serial time points between 6 and 36 h posttrans-
fection and resuspended in 55 l of ice-cold lysis buffer (10 mM HEPES, 10 mM
NaCl, 3 mM CaCl
, 7 mM MgCl
, 1 mM EDTA, 0.5% NP-40). Cytoplasmic
lysates were harvested by centrifugation at 9,300 ⫻ g for 2 min and subjected to
a Gag enzyme-linked immunosorbent assay (ELISA) or a luciferase assay. For
reporter gene assays and LMB sensitivity experiments, 293 cells (3 ⫻ 10
60-mm-diameter plates were transfected with 1 g of a reporter plasmid using a
protocol (7). LMB was added 24 h posttransfection, and cells were
harvested 24 h later.
HIV-1 Gag levels were quantiﬁed by Gag ELISA (Coulter Corp.) and nor-
malized to ﬁreﬂy Luc activity expressed from cotransfected pGL3 (Promega). In
DNA transfections that used ﬁreﬂy luc as the reporter gene, pRL-CMV (Pro-
mega) was cotransfected and Renilla luciferase activity was used to standardize
transfection efﬁciency. Luc or Renilla luciferase assays were performed with 10 l
of lysate and 100 l of substrate (Promega), and enzyme activity was quantiﬁed
in a Lumicount luminometer (Packard).
RNA preparation. Transfected 293 cells from three 10-cm-diameter plates that
were treated with LMB at 0 or 2.5 ng/ml were harvested in PBS and lysed on ice
for 15 min in a solution containing 10 mM Tris (pH 8.3), 150 mM NaCl, 1.5 mM
, and ⫺0.5% NP-40, and nuclei were removed by centrifugation at 10,000
⫻ g for 10 min. The cytoplasmic supernatant was recentrifuged at 10,000 ⫻ g for
10 min, and the clariﬁed extract was mixed with Tri-Reagent LS (Molecular
Research). The nuclear pellets were washed twice in PBS and mixed with Tri-
Reagent (Molecular Research). The RNAs were extracted in accordance with
the manufacturer’s protocol and treated with DNase I.
Cytoplasmic factors are insufﬁcient for SNV RU5 transla-
tional enhancement. We compared reporter gene activity from
293 cells transfected either with reporter plasmid or liposomes
containing in vitro-transcribed capped and polyadenylated re-
porter RNA. The RNA transfection assay has been used to
assess reporter protein synthesis independently of de novo
transcription, RNA processing, and nuclear export and pro-
vides an in vivo approach by which to determine whether
cytoplasmic factors are sufﬁcient for translational enhance-
ment by SNV RU5 (43).
Consistent with our published data, results of DNA trans-
fection assays in 293 cells indicate that SNV RU5 in the sense
orientation confers Rev/RRE-independent expression of in-
tron-containing gag RNA (Table 1, pYW100 and pYW208)
(7). Furthermore, SNV RU5 augments expression of nonviral
luc reporter RNA (compare pTR103 and pTR105) (35). Efﬁ-
cient Gag production is not observed from Gag reporter plas-
mids that lack SNV RU5 (pYW205, pYW99, and pSVgagpol-
rre) or contain antisense SNV RU5 (pTR147 and pYW207) or
substitution mutant SNV RU5 (pYW233). As expected, co-
transfected pRev activates expression from the RRE-contain-
ing plasmids (pSVgagpolrre and pTR155).
Results obtained with the synthetic capped and polyadenyl-
ated gag reporter RNAs indicate that SNV RU5 gag RNA is
efﬁciently utilized as template mRNA for Gag protein synthe-
sis (Fig. 1, 19 ⫾ 0.1 ng/ml, normalized to 1). However, unlike
results obtained with the reporter plasmids, similar levels of
Gag are synthesized from gag reporter RNAs that contain
deleted, antisense, or substitution mutant SNV RU5. Luc ac-
tivity also remained similar upon transfection with luc RNA
that contained sense, antisense, or deleted SNV RU5 (44,000
VOL. 76, 2002 Rev/RRE DISRUPTS SNV RU5 TRANSLATION ENHANCEMENT 3293
⫾ 13,000 relative light units [RLU], normalized to 1). The lack
of SNV RU5 stimulation was sustained when dilutions of the
reporter RNAs were transfected and when shorter incubation
periods were evaluated, indicating that the RNAs were not
used at saturating levels and were able to titrate SNV RU5-
interactive proteins (data not shown). The results indicate that
SNV RU5 does not confer a positive effect on the translation
of either gag or luc reporter transcripts that are introduced
directly into the cytoplasm. The data imply that interaction
between SNV RU5 RNA and a nuclear factor(s) is necessary
for the positive effect of SNV RU5. An alternative explanation
is that the structural conformation of SNV RU5 is not reca-
pitulated in liposomes. However, Stoneley et al. observed that
the human rhinovirus type 2 IRES remains functional in the
liposome-mediated RNA transfection assay (43).
Because a cell-free assay would be a useful tool with which
to investigate SNV RU5-interactive proteins, we also investi-
gated the utility of in vitro translation assays. RRL pro-
grammed with sense SNV RU5 gag transcripts produced sig-
niﬁcant Gag protein (Fig. 1, 100 ⫾ 8.7 ng/ml, normalized to 1).
Gag production was sustained or increased fourfold in RRL
programmed with an antisense SNV RU5 gag or deletion-
containing SNV RU5 gag transcript, respectively. Analysis of
luc transcripts identiﬁed that RRL programmed with sense
FIG. 1. Protein synthesis from capped and polyadenylated reporter transcripts reveals that cytoplasmic proteins are insufﬁcient for translational
enhancement by SNV RU5. Shown are representative transcripts used to program three translation systems and their designations, structures, and
relative protein-synthetic activities. RNA transfection of 293 cells was performed with 2.5 g of transcript and Lipofectamine. RRL and 293 cell
lysates were programmed with 400 ng of reporter transcript. In each assay system, luc or gag reporter transcripts were used reciprocally to
standardize minor differences in RNA transfection efﬁciency. Reporter proteins were measured by a Gag ELISA or a Luc enzymatic assay. ND,
TABLE 1. Reporter protein synthesis
Plasmid Promoter RU5 RRE Reporter gene Gag concn (ng/ml) Luc activity (RLU)
pYW100 SNV Sense Absent gag 41.2 ⫾ 1.8 NA
pYW208 CMV Sense Absent gag 18.0 ⫾ 1.1 NA
pYW205 SNV Absent Absent gag 5.0 ⫾ 0.9 NA
pYW99 CMV Absent Absent gag ⬍MD
pTR147 SNV Antisense Absent gag ⬍MD NA
pYW207 CMV Antisense Absent gag ⬍MD NA
pYW233 SNV Mutant Absent gag ⬍MD NA
pTR155 SNV Antisense Present gag ⬍MD NA
pTR155⫹Rev SNV Antisense Present gag 210.0 ⫾ 81.2 NA
Absent Present gag ⬍MD NA
pSVgagpolrre⫹Rev SV40 Absent Present gag 160.5 ⫾ 8.7 NA
pTR103 SNV Sense Absent luc NA 42 ⫾ 2.5
pTR105 SNV Absent Absent luc NA 8 ⫾ 0.1
⬍MD, less than the minimum detectable, 0.02 ng/ml.
NA, not applicable.
SV40, simian virus 40.
3294 DANGEL ET AL. J. VIROL.
SNV RU5 luc exhibited Luc activity (8,300 ⫾ 1,700 RLU,
normalized to 1), while antisense SNV RU5 luc transcripts
increased Luc activity 2-fold and deletion of SNV RU5 in-
creased Luc activity 11-fold. The inhibitory effect of SNV RU5
on protein synthesis in RRL is likely attributable to structural
barriers in SNV RU5 RNA. Structural barriers in HIV-1 RU5
and other 5⬘ UTRs have been previously shown to inhibit
protein synthesis in RRL (18, 22, 29). We also performed in
vitro translation assays with 293 cell cytoplasmic lysates be-
cause 293 cells had been used to characterize SNV RU5 in the
DNA transfection assays. The extracts were veriﬁed to be
translationally competent by [
into nascent polypeptide chains (data not shown) and by pro-
gramming with reporter transcripts and conﬁrmation of re-
porter protein synthesis (Fig. 1). Gag production in response to
a sense SNV RU5 gag or a mutant SNV RU5 gag transcript was
the same (50 ⫾ 7 ng/ml, normalized to 1). The lack of SNV
RU5 stimulation in vitro was sustained in the RRL or 293 cell
extracts when dilutions of reporter RNA were tested and when
shorter incubation periods were evaluated, indicating that the
RNAs were not used at saturating levels. The data from these
in vitro translation systems are consistent with the RNA trans-
fection results and indicate that the positive effect of SNV RU5
is not recapitulated in these cytoplasmic extracts. The data
imply that nuclear interactions are necessary for translational
enhancement by SNV RU5. An alternative explanation is that
these conventional in vitro translation extracts are deﬁcient in
faithful regulation of translation initiation.
LMB does not inhibit cytoplasmic accumulation of SNV
RU5 gag RNA. LMB inhibits activation of nuclear export by
Rev/RRE by alkylation of CRM1 at Cys-529 and prevention of
leucine-rich NES-mediated nuclear export (17, 33, 44). DNA
transfection assays were used to investigate whether or not
nuclear export of SNV RU5 gag reporter RNA is sensitive to
LMB. Triplicate cultures of transfected 293 cells were treated
with various concentrations of LMB for 24 h posttransfection,
and cell-associated reporter proteins were quantiﬁed. Consis-
tent with previous reports, LMB severely reduced Gag produc-
tion in cells cotransfected with pRev and RRE-containing plas-
mid pSVgagpolrre (lacks SNV RU5) or pTR155 (contains
antisense SNV RU5) (Fig. 2). By contrast, Gag production
from pYW100 (contains SNV RU5) was not inhibited by LMB
and is reproducibly enhanced in response to LMB. Similar
responses were observed when SNV RU5 was positioned
downstream of the SNV U3 promoter (pYW100) or the cyto-
megalovirus (CMV) immediate-early promoter (pYW208)
(Fig. 3). Deletion of SNV RU5 (pYW205), which reduces Gag
production to low but detectable levels, eliminated the LMB
enhancement of Gag production. The data indicate that SNV
RU5 confers LMB-enhanced expression in a promoter-inde-
The lack of LMB inhibition of Gag production from SNV
RU5 gag reporter RNA suggests that the RNA accesses a
nuclear export pathway that is distinct from the leucine-rich
NES/CRM1-mediated pathway utilized by Rev/RRE. To eval-
uate the effect of LMB on cytoplasmic accumulation of SNV
RU5 gag RNA, RPAs were performed on nuclear and cyto-
plasmic RNA from transfected 293 cells treated with LMB at
0 or 2.5 ng/ml. The uniformly labeled HIV antisense RNA
probe is complementary to the 5⬘ UTR and distinguishes un-
spliced gag mRNA and spliced versions of the primary tran-
script (Fig. 4A) (7). LMB treatment of cells transfected with
pYW100 increased the cytoplasmic accumulation of SNV RU5
gag threefold (Fig. 4B and C). The increase in cytoplasmic
SNV RU5 gag RNA is proportional to the fourfold increase in
Gag protein observed in response to LMB (Table 2). Cytoplas-
mic accumulation of spliced SNV RU5 transcripts displayed a
similar 2.6-fold increase, indicating that both spliced and intron-
containing versions of SNV RU5-containing RNA are affected by
LMB. In contrast, cells transfected with the RRE-containing re-
porter pTR155 and pRev exhibited a reduction in the cytoplasmic
accumulation of RRE gag RNA and Gag production by a factor
of 0.6. These data indicate that SNV RU5 provides both spliced
and intron-containing RNAs access to a nuclear export pathway
FIG. 2. Effect of LMB on Gag production reveals that SNV RU5
functions independently of CRM1/leucine-rich NES. Results of three
to ﬁve replicate transfections of 293 cells with the indicated gag re-
porter plasmids are shown. Reporter proteins were measured 24 h
posttreatment with the indicated concentrations of LMB. Gag produc-
tion is presented relative to that of the mock-treated control.
FIG. 3. LMB dose-response curves indicate that SNV RU5 confers
LMB enhancement in a promoter-independent manner. Results of
three replicate transfections of 293 cells with the indicated reporter
plasmids are shown. Cell-associated reporter proteins were measured
24 h posttreatment with the indicated concentrations of LMB. Pre-
sented is Gag protein production in response to the indicated gag
reporter plasmid relative to that of the mock-treated control.
VOL. 76, 2002 Rev/RRE DISRUPTS SNV RU5 TRANSLATION ENHANCEMENT 3295
that is distinct from the NES-CRM1-mediated Rev/RRE path-
way. A possible explanation for the LMB enhancement of gag
RNA expression conferred by SNV RU5 is that CRM1 inactiva-
tion alters the availability of a rate-limiting SNV RU5 nuclear
export or RNA stability cofactor(s).
The combination of SNV RU5 and Rev/RRE augments Gag
expression. We sought to evaluate whether or not the combi-
nation of SNV RU5 and Rev/RRE on a single RNA would
synergistically augment gag gene expression. The RRE was
introduced into pYW100 (contains RU5) and pYW205 (lacks
FIG. 4. RPA of cytoplasmic and nuclear RNAs from transfected 293 cells with or without 24 h of LMB treatment reveals that LMB augments
cytoplasmic accumulation of SNV RU5 gag RNA. (A) Relationships among the gag reporter plasmid, an antisense HIV-1 5⬘ UTR RNA probe,
and protected unspliced and spliced transcripts with sizes indicated. ss, splice site; nt, nucleotides. (B) RPA and PhosphorImager analysis of
cytoplasmic (30 g) and nuclear (15 g) RNAs. Labels indicate the reporter plasmid, LMB treatment, and protected transcript. (C) Northern blot
analysis of 10-g aliquots of the RNA samples hybridized to an actin probe.
TABLE 2. Effects of LMB on protein and RNA levels
Minus LMB Plus LMB
Nuclear RNA Cytoplasmic RNA
Unspliced 363 5,028 794 1,676 6,902 3,334 3.1
27,774 42,012 NA 22,897 89,606 2.6
pTR155 ⫹ pRev
Unspliced 1,442 5,409 9,691 658 5,074 5,581 0.6
Spliced NA 3,310 6,214 NA 3,306 4,946 0.8
Gag protein levels were measured by ELISA.
RNA levels were determined by RNase protection assay and are expressed in PhosphorImager units and normalized to the actin RNA signal.
Ratio of cytoplasmic RNA to nuclear RNA in the presence and absence of LMB.
NA, not applicable.
3296 DANGEL ET AL. J. VIROL.
RU5), and Gag production was measured in the presence or
absence of Rev. The introduction of RRE had little effect on
Gag production in the absence of Rev (Table 3). As expected,
cotransfection of Rev transactivated Gag production from
pYW100RRE and from pYW205RRE). Notably, the magni-
tude of the increase in Gag production was smaller in the
presence of SNV RU5 than in the absence of SNV RU5
(compare pYW100RRE [34-fold] and pYW205RRE [95-
fold]). To address the possibility that the lower fold transacti-
vation is attributable to a maximum threshold of Gag produc-
tion from pYW100RRE plus Rev, dose-response curves were
generated with twofold molar increases of cotransfected pRev.
Gag production from pYW100RRE, pYW205RRE, and
pTR155 increased progressively in response to increasing pRev
and demonstrated that Gag production had not reached a
maximum threshold (data not shown). Furthermore,
pYW100RRE RNA continued to exhibit a smaller magnitude
of Rev transactivation. An alternative explanation for the
lower fold Rev transactivation for pYW100RRE is that Rev/
RRE disrupts translational enhancement by SNV RU5. Con-
sistent with this possibility, the positive effect of SNV RU5
was reduced in the presence of Rev. The positive effect of
SNV RU5 was 7.5-fold in the absence of Rev (compare
pYW100RRE [30 ⫾ 1.5 ng/ml] and pYW205RRE [4 ⫾ 0.8
ng/ml]) and was reduced to 2.7-fold in the presence of Rev
(compare pYW100RRE plus pRev [1,030 ⫾ 350 ng/ml] and
pYW205RRE plus pRev [380 ⫾ 37 ng/ml]).
NES/CRM1-dependent Rev/RRE nuclear export pathway is
dominant and impedes translational enhancement by SNV
RU5. LMB was used to determine whether or not Rev/RRE
can divert SNV RU5 gag RNA to the LMB-inhibited CRM1/
NES-dependent pathway. In the absence of Rev, pYW100RRE
exhibits LMB-enhanced Gag production (Fig. 5), similar to
those from pYW100 (Fig. 2A). Gag levels from pYW100RRE
increased to 190% ⫾ 8%, indicating that RRE does not disrupt
LMB enhancement by SNV RU5. However, in the presence of
Rev, Gag production from pYW100RRE was inhibited by
LMB (Fig. 5). Gag production was reduced to a level similar to
that of pYW205RRE plus pRev (lacks RU5). The shift of
pYW100RRE RNA from LMB enhancement in the absence of
Rev to LMB inhibition in the presence of Rev indicates that
SNV RU5 and Rev/RRE compete for posttranscriptional con-
trol of gag RNA. Rev/RRE appears to sequester the SNV RU5
transcripts to the LMB-sensitive NES/CRM1-mediated nu-
clear export pathway.
Quantitative RNA and protein analysis was used to deter-
mine whether or not sequestration to the Rev/RRE pathway
abrogates SNV RU5 translational enhancement. The positive
effect of Rev on cytoplasmic accumulation of the gag RNA was
similar for pYW100RRE (contains SNV RU5) and pTR155
(antisense SNV RU5) RNAs, 4.5-fold and 2.9-fold, respec-
tively (Fig. 6A; Table 4). Consistent with the results in Table 3,
Rev transactivated Gag production from both reporters.
Again, the magnitude of the increase was smaller in the pres-
ence of SNV RU5 than in the absence of SNV RU5 (14-fold
versus 42-fold). The translational efﬁciency of the gag RNAs
remained similar for pYW100RRE and pTR155 in the pres-
ence of Rev, 0.26 and 0.30, respectively. However, in the ab-
sence of Rev, the translational efﬁciency of pYW100RRE was
greater by 2.6-fold (Fig. 6B). These results indicate that SNV
RU5 and Rev/RRE do not function in a synergistic manner to
increase the cytoplasmic expression of gag RNA. The augmen-
tation of translational efﬁciency observed with RU5 alone
(compare pTR155 [1.0-fold] and pYW100RRE [2.6-fold]) is
abrogated upon Rev/RRE and RU5 combination (compare
pTR155 plus Rev [4.3-fold] and pYW100RRE plus Rev [3.7-
fold]) (Fig. 6B). Similar results were observed in replicate ex-
periments with pYW205RRE (lacks RU5) and pYW100RRE
(data not shown). The elimination of the positive effect of SNV
RU5 by Rev/RRE correlates with sequestration to the LMB-
inhibited CRM1/NES nuclear export pathway. A possible ex-
planation is that SNV RU5, but not Rev/RRE, recruits a nu-
clear factor(s) that is necessary to program the RU5 gag RNA
for translational enhancement in the cytoplasm.
Our results uncover a functional linkage between the SNV
RU5 nuclear export pathway and translational enhancement of
intron-containing retroviral mRNA. Results of RNA transfec-
tion assays with synthetic gag and luc RNAs that are capped
and polyadenylated indicate that cytoplasmic factors are insuf-
ﬁcient for translational enhancement by SNV RU5. We ob-
served that gag RNA introduced into the cytoplasm by RNA
transfection is functional template mRNA for protein synthe-
sis. Our data corroborate extensive studies with reporter plas-
mids and HIV provirus indicating that the Rev/RRE depen-
FIG. 5. LMB dose-response curves indicate that LMB enhance-
ment of SNV RU5 gag RNA is mitigated by Rev/RRE. 293 cells
were transfected with the indicated gag reporter plasmid, and cell-
associated Gag levels are presented relative to that of the mock-
TABLE 3. Increase in Gag production due to Rev
Gag concn (ng/ml)
Minus Rev Plus Rev
pYW100 Present 50 ⫾ 4 (12.5)
pYW100RRE Present 30 ⫾ 1.5 (7.5) 1,030 ⫾ 95 (260) 34
pYW205 Absent 3 ⫾ 0.7 (0.75) NA NA
pYW205RRE Absent 4 ⫾ 0.8 (1.0) 380 ⫾ 37 (95) 95
Increase in response to Rev.
In parentheses is the level relative to that obtained with pYW205RRE minus
NA, not applicable.
VOL. 76, 2002 Rev/RRE DISRUPTS SNV RU5 TRANSLATION ENHANCEMENT 3297
dence of gag RNA is attributable to derepression of nuclear
retention (6, 9a, 14, 26a, 37a). Consistent with previous studies,
our in vitro translation data obtained with RRL and 293 cell
lysates indicate that neither Rev/RRE nor SNV RU5 is nec-
essary for Gag protein synthesis in cytoplasmic extracts (12).
The positive effect of SNV RU5 was not recapitulated in the in
vitro translation extracts. This negative result is consistent with
a role for nuclear factors or the notion that translational reg-
ulation is not faithfully reconstituted in these cell extracts.
Recently, a protocol was developed with HeLa cells for the
FIG. 6. RPA of nuclear and cytoplasmic RNAs from transfected 293 cells to quantify cytoplasmic RNA accumulation in response to Rev/RRE
and SNV RU5. Cytoplasmic (30 g) and nuclear (15 g) RNAs were protected with a
P-labeled antisense HIV-1 5⬘ UTR RNA probe and a
GAPDH RNA probe and were visualized by PhosphorImager analysis. Labels indicate the protected transcripts. (A) RNA expressed from
pYW100RRE or pTR155 in the absence or presence of Rev. (B) Translational efﬁciency is presented relative to that of pTR155. The ratio of Gag
protein to cytoplasmic RNA is from Table 4.
TABLE 4. Effects of Rev on protein and RNA levels
Minus Rev Plus Rev
Cytoplasmic RNA TE
Nuclear RNA Cytoplasmic RNA TE
Unspliced 219 11,599 1,194 0.18 2,985 (14)
24,522 11,388 0.26 4.5
14,236 10,675 NA NA 12,405 10,417 NA 1.1
Unspliced 33 1,399 452 0.07 1,391 (42) 5,037 4,819 0.30 2.9
Spliced NA 858 1,395 NA NA 1,840 2,777 NA 0.9
RNA levels were determined by RNase protection assay and are expressed in PhosphorImager units and normalized to the GAPDH signal.
TE, translational efﬁciency (ratio of Gag protein production to cytoplasmic RNA level).
Ratio of cytoplasmic RNA to nuclear RNA in the presence and absence of Rev.
Fold increase in response to Rev.
NA, not applicable.
3298 DANGEL ET AL. J. VIROL.
preparation of in vitro translation extracts that faithfully reca-
pitulate the synergistic interplay between the 5⬘ cap structure
and the poly(A) tail during translation initiation (4). Recapit-
ulation of this aspect of translational regulation may be nec-
essary to execute SNV RU5 translational enhancement in
LMB was used as a tool with which to determine that SNV
RU5-containing RNAs achieve nuclear export independently
of the leucine-rich NES/CRM1-independent nuclear export
pathway utilized by Rev/RRE. Recently, similar CRM1-inde-
pendent RNA export of intron-containing RNA was reported
for the Rous sarcoma virus direct repeat (DR) element, the
MPMV constitutive transport element, and the hepatitis B
virus posttranscriptional regulatory element (PRE) (33, 34,
45). SNV RU5 is distinct in affecting both intron-containing
and intron-lacking reporter RNAs. At high concentrations of
LMB (5 to 10 nM), DR element- and PRE-mediated gene
expression is increased (33, 34), which is reminiscent of our
results obtained with SNV RU5. Although the DR element
and the PRE do not share with SNV RU5 a direct effect on
translational efﬁciency, the DR element augments other as-
pects of cytoplasmic expression, including RNA stability, RNA
packaging, and efﬁcient virus assembly (2, 31, 32, 39, 40, 42).
SNV RU5 and the DR element may recruit common cellular
factors that provide access to a distinct CRM1-independent
nucleocytoplasmic export pathway that targets the RNA for
cytoplasmic localization at particular subcellular microenviron-
ments, active translation centers in the case of SNV RU5, and
virus assembly sites in the case of the DR element.
The combination of SNV RU5 and Rev/RRE on a single
RNA provided a unique tool with which to investigate the
relationship between utilization of a particular nuclear export
pathway and translational enhancement by SNV RU5. First,
our data demonstrate that Rev/RRE is signiﬁcantly more po-
tent than SNV RU5 in its magnitude of posttranscriptional
activation. Second, the combination of Rev/RRE and SNV
RU5 increases posttranscriptional gene expression in a less-
than-additive manner. The small positive effect of the combi-
nation of SNV RU5 and Rev/RRE correlates with a small
increase in cytoplasmic RNA accumulation of gag RNA and
may be attributable to an increase in the stability or export of
the RNA. Third, in the presence of Rev/RRE, LMB-inhibited
nuclear export of RU5 gag-RRE RNA is observed to function
dominantly over the LMB-enhanced pathway. Fourth, seques-
tration of SNV RU5 gag-RRE RNA to the LMB-enhanced
pathway correlated with abrogation of translational enhance-
ment by SNV RU5. The possibility that the translational efﬁ-
ciency of the reporter RNA had reached a maximum level was
eliminated by the observation that the gag RNA was competent
for increased amounts of Gag protein synthesis when Rev was
overexpressed. Our results support the model in which inter-
action between SNV RU5 and a nuclear factor(s) is necessary
for SNV RU5-mediated translational enhancement.
We speculate on two possible roles of nuclear factors in SNV
RU5-mediated translational enhancement. First, SNV RU5
may interact with a nuclear factor that is, or recruits, a rate-
limiting translation initiation factor. An attractive candidate is
eIF4E because eIF4E shuttles between the nucleus and cyto-
plasm and is the rate-limiting initiation factor for cap-depen-
dent translation (13). In a second model, SNV RU5 interacts
with a cellular protein that resembles Rev in the ability to
provide nuclear export to a particular RNA export pathway
and direct polysome association. This scenario is similar to the
recent suggestion that R of HFV directs HFV Gag translation
by delivery of the RNA to a translationally active environment
in the cytoplasm (37). While Rev functions cotranscriptionally
(23) to activate nuclear export by the leucine-rich NES/CRM1-
dependent pathway and selectively drives intron-containing gag
RNA along a particular cytoplasmic circuit, the hypothetical
Rev-like factor would bind SNV RU5 and facilitate export and
polysome association along a different cytoplasmic circuit. The
dominance of Rev/RRE may be attributable to cotranscrip-
tional interaction of Rev/RRE and host proteins at a point
upstream of interaction between SNV RU5 and an RU5-in-
teractive protein(s). We note that SNV RU5 confers LMB en-
hancement in the context of either the SNV U3 or CMV
immediate-early promoter/enhancer, indicating that recruit-
ment of RU5-interactive factors is not strictly dependent on
cotranscriptional recruitment by the SNV promoter. SNV,
HFV, and MPMV are divergent retroviruses, but they share 5⬘
RNA elements that may interact with a common nuclear fac-
tor(s) that programs the intron-containing RNA for productive
We thank Minoru Yoshida (University of Tokyo, Tokyo, Japan) for
the gift of LMB, Paul Copeland (Cleveland Clinic, Cleveland, Ohio)
for expert advice on preparation of 293 cell translation lysates, and
Jennifer Frey for plasmid construction. We are grateful to Michael
Lairmore for comments on the manuscript and Patrick Green for
This work was supported by grants from the National Institute of
Allergy and Infectious Diseases (R29AI40851) and the National Can-
cer Institute (P30CA16058), Bethesda, Md.
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