Published online 10 April 2007 Nucleic Acids Research, 2007, Vol. 35, No. 8 2629–2642
doi:10.1093/nar/gkm124
RNA helicase A interacts with divergent lymphotropic
retroviruses and promotes translation of human
T-cell leukemia virus type 1
Cheryl Bolinger1,4, Alper Yilmaz1,4, Tiffiney Roberts Hartman1,2, Melinda Butsch Kovacic1,
Soledad Fernandez5,6, Jianxin Ye1,4, Mary Forget1,4, Patrick L. Green1,2,3,4,6 and
Kathleen Boris-Lawrie1,2,3,4,6,*
1Center for Retrovirus Research, 2Department of Veterinary Biosciences and 3Department of Molecular
Virology, Immunology & Medical Genetics, 4Molecular, Cellular & Developmental Biology Graduate Program,
5Center for Biostatistics and 6Comprehensive Cancer Center, The Ohio State University, Columbus,
OH 43210-1093, USA
Received September 15, 2006; Revised January 25, 2007; Accepted February 14, 2007
ABSTRACT
The 50 untranslated region (UTR) of retroviruses
contain structured replication motifs that impose
barriers to efficient ribosome scanning. Two RNA
structural motifs that facilitate efficient translation
initiation despite a complex 50 UTR are internal
ribosome entry site (IRES) and 50 proximal post-
transcriptional control element (PCE). Here, strin-
gent RNA and protein analyses determined the 50
UTR of spleen necrosis virus (SNV), reticuloen-
dotheliosis virus A (REV-A) and human T-cell
leukemia virus type 1 (HTLV-1) exhibit PCE activity,
but not IRES activity. Assessment of SNV translation
initiation in the natural context of the provirus
determined that SNV is reliant on a cap-dependent
initiation mechanism. Experiments with siRNAs
identified that REV-A and HTLV-1 PCE modulate
post-transcriptional gene expression through inter-
action with host RNA helicase A (RHA). Analysis of
hybrid SNV/HTLV-1 proviruses determined SNV PCE
facilitates Rex/Rex responsive element-indepen-
dent Gag production and interaction with RHA is
necessary. Ribosomal profile analyses determined
that RHA is necessary for polysome association of
HTLV-1 gag and provide direct evidence that RHA is
necessary for efficient HTLV-1 replication. We
conclude that PCE/RHA is an important translation
regulatory axis of multiple lymphotropic retro-
viruses. We speculate divergent retroviruses have
evolved a convergent RNA–protein interaction
to modulate translation of their highly structured
mRNA.
INTRODUCTION
Retrovirus structural and enzymatic proteins are synthe-
sized from mRNA transcript that contains a complex 50
untranslated region (UTR) that inhibits ribosome scan-
ning (1–4). The process used to circumvent this barrier to
translation initiation is an issue that remains to be fully
elucidated. Translation initiation is the rate-limiting step
in protein synthesis and the primary target for transla-
tional control (5). The majority of eukaryotic mRNAs use
a cap-dependent mode of translation initiation in which
the multicomponent eIF4F complex binds the 50 7mpppG
cap of mRNA and is joined by the small ribosomal
subunit (5,6). Once assembled, the initiation complex
scans the mRNA until an AUG or, in some cases CUG,
in appropriate Kozak consensus is located. The large
ribosomal subunit then joins the complex and the
elongation phase begins. A 50 UTR greater than 100 nt
in length or with structural motifs with free energy of
4
50 kcal/mol has been shown to block efficient ribosome
scanning and significantly dampen the efficiency of
translation initiation (1,7). The UTR of retroviruses is
typically greater than 200 nt in length and contain a
collection of cis-acting replication motifs with free energy
greater than
50 kcal/mol (8). These motifs include the
packaging signal and primer-binding site, which are stem
loop structures that perform essential functions during
particle assembly and reverse transcription, respectively.
In order to ensure translation of the viral transcript,
retroviruses require a specialized translational control
mechanism to overcome structural barriers to cap-
dependent ribosome scanning. Two viral RNA elements
that facilitate translation despite a long and highly
structured 50 UTR are: (1) internal ribosome entry site
(IRES); and (2) post-transcriptional control element
(PCE). Both viral RNA elements operate by recruiting
*To whom correspondence should be addressed. Tel: þ1-614-292-1392; Fax: þ1-614-292-6473; Email: boris-lawrie.1@osu.edu

2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

host effector proteins and have been shown to have a
homolog in cellular genes.
The IRES is an RNA structural motif originally
identified in the picornaviruses encephalomyocarditis
virus (EMCV) (9–11) and poliovirus (12), and more
recently in selected cellular mRNAs including BIP (13),
eIF4G (14), c-Myc (15,16), DAP5 (17), Apaf-1 (18),
BAG-1 (19,20), c-IAP1 (21), FGF2 (22,23) and BCL-2
(24). Picornaviruses lack a 50 cap and contain a 50
UTR 750 nt in length that is comprised of structural
motifs, oligopyrimidine tracts and cryptic AUG and CUG
codons upstream of the authentic initiation codon.
Features of this complex 50 UTR serve as a recognition
site for a modified pre-initiation complex that delivers the
small ribosomal subunit to an appropriate initiation
codon independently of cap binding and ribosome
scanning. Generally, picornaviruses encode proteins that
modify the eIF4F complex and efficiently shutdown
cap-dependent host translation initiation. Under such
conditions, cap-independent translation initiation at the
viral IRES is favored (25).
Unlike picornaviruses, retroviral mRNAs are capped,
polyadenylated, and in some cases lack upstream AUGs.
They also generally contain AUGs in appropriate Kozak
consensus (8). These features are consistent with transla-
tion initiation by a cap-dependent mechanism. However,
the structured replication motifs between the 50 cap and
distal translation start codon of retroviral RNAs have
been directly demonstrated to inhibit efficient cap-
dependent ribosome scanning and impede translation
initiation at the downstream open reading frame (2–4).
A growing collection of retroviruses have been identified
to contain a 50 terminal PCE that functions in conjunction
with cellular protein to facilitate translation of retroviral
genes. PCE activity has been identified in three lympho-
tropic retroviruses: avian SNV, simian Mason–Pfizer
monkey virus (MPMV) and human foamy virus (26–28).
Results derived from the retrovirus model system led to
the first identification of PCE activity in a cellular
gene, junD (29). Located at the 50 RNA terminus, the
PCE is a 160 nt, orientation-dependent redundant
stem-loop structure that was identified by its ability to
facilitate Rev/RRE-independent expression of intron-
containing HIV-1 gag reporter RNA (26,28,30). RNA
co-immunoprecipitation and biochemical analysis identi-
fied that structural features of PCE are selectively
recognized by the cellular DEaH box protein RNA
helicase A (RHA) (29). PCE–RHA interaction is neces-
sary for efficient translation initiation and functions by
facilitating polyribosome association (26,29,31).
Results of bicistronic reporter assays determined that
the 160-nt SNV PCE does not confer IRES activity (31).
However, the potential for IRES activity in the distal 50
UTR of SNV remained to be determined. SNV shares
90% sequence homology with reticuloendotheliosis virus
A (REV-A) and IRES activity has been identified in distal
50 UTR sequences of REV-A (32). This IRES activity was
identified in the context of murine leukemia virus (MLV)-
based bicistronic vectors containing various REV-A
50 UTR segments. The presence of the complete REV-A
50 UTR (sequencesþ 1-580) upstream of the neomycin
phosphotransferase reporter gene correlated with
expression of G418 resistance gene (32). The minimum
sequence determinant for expression of G418 resistance
was mapped to a 129-nt distal segment of the 50 UTR
(452–580).
Bicistronic reporter assays have also detected IRES
activity in the 50 UTR of human T-cell leukemia virus
type 1 (HTLV-1) (33,34), Harvey murine sarcoma virus
(35), MLV (36–38), Rous sarcoma virus (RSV) (39),
simian immunodeficiency virus (SIV) (40), human immu-
nodeficiency virus 1 (HIV-1) (41) and in the gag open
reading frame of HIV-1 (42) and HIV-2 (43). Internal
translation initiation in the natural context of the
virus genome has yet-to-be characterized for REV-A or
HTLV-1 and remains controversial in HIV-1 (2–4,8).
The goal of this study was comparative analysis
of IRES and PCE activity within the 50 UTR of SNV,
REV-A and HTLV-1. Our results implicate PCE in
association with RHA is a key translation regulatory
axis of multiple retroviruses.
MATERIALS AND METHODS
Plasmid construction
Bicistronic reporter plasmids containing SNV sequences
were constructed using polymerase chain reaction (PCR)
of the designated sequences from SNV provirus clone
pPB101 (44) and primers containing EcoRI, NcoI and
XbaI sites and inserted into the EcoRI and NcoI sites
of pTR250 (31). The SNV UTR-luc and polio IRES-luc
cassettes were excised with XbaI and ligated at the XbaI
site in pRL-CMV (Promega, Madison, WI) to create
pSNV/1-591, pSNV/260-591, pSNV/393-591 and pPolio.
pNoIRESfs is identical to previously described pRenLuc
(45). The bicistronic reporter plasmids containing
HTLV-1 50 UTR sequencesþ 1-263 was created by PCR
of HTLV-1 strain ACH with primer-containing SalI sites
and inserted in SalI sites of intermediate bicistronic
plasmid pCG201. pREV-A was created by PCR amplifi-
cation of REV-A provirus pSW253 50 UTR sequencesþ 1-
580 using primers containing SalI and inserted into SalI
sites of pCG201. To construct pSNVgagluc, pPB101 SNV
pol was replaced with F-luc in three steps. pPB101 was
digested with SmaI and religated to delete sequences
2964–5632. PCR-based site-directed mutagenesis was used
to eliminate the pol stop codon and introduce a NcoI site.
This plasmid, pMB109, was digested with NcoI and
SmaI and ligated in-frame with the luc ORF from pGL3
(Promega, Madison, WI) to create pSNVgagluc. The
previously described plasmids pYW100 and pYW205 (26)
were used to generate REV-A derivatives. The REV-A
LTR or U3 region in a PCR product of pSW253
containing terminal NdeI and BglII was inserted into
NdeI and BamHI sites of pYW100 or pYW205 to
generate pREV-A100 and pREV-A205. Plasmids contain-
ing HTLV-2 (46), BLV (47), EIAV (48) and FeLV (49)
LTR regions were constructed similar to pREV-A100.
pCG211 was constructed by digestion of pREV-A100 and
pYW100 with NdeI and AvaI and subcloning of SNV U3
from pYW100 into pREV-A100. pMPMV100 is the
2630 Nucleic Acids Research, 2007, Vol. 35, No. 8

previously described pMPMV (28). Generation of Rex-
deficient HTLV-1 proviral clone HTLVRex- was
described (50). To construct wt/PCE and Rex-/PCE,
EcoRV and BamHI sites were generated in the R region
of wild-type and the Rex-deficient HTLV-1ACH (26) by
site-directed mutagenesis (QuickchangeTM, Stratagene),
and used for insertion of SNV PCE inserted between
EcoRV and BamHI. The plasmid constructions were
confirmed by DNA sequence analysis.
Transfections and protein analysis
Cultures of 293 and COS cells were grown to 80%
confluency in Dulbecco’s modified eagle medium
(Invitrogen) with 10% FBS (Invitrogen) and 1% anti-
biotic (Gibco) prior to transfection. DNA transfection
was carried out with a 3:1 FuGene:DNA ratio following
the manufacturers protocol (Roche). RNA transfections
were performed using Transmessenger transfection
reagent (Qiagen). Cells were pelleted by centrifugation at
3500 r.p.m., resuspended in NP40 lysis buffer, iced for
10min, then centrifuged at 12000 r.p.m. at 48C for 10min
to collect proteins as previously described (51). Luciferase
activity was measured with Luciferase assay system
(Promega) or Dual Luciferase Assay system (Promega)
following the manufacturer’s protocol quantified on a
Lumicount luminometer (Packard). HIV-1 Gag levels
were quantified by Gag enzyme-linked immunosorbent
assay (ELISA) (Coulter Corp.) and HTLV Gag levels
were quantified by ELISA (Zeptometrix).
For western blot analysis, proteins were subjected
to 10% SDS-PAGE and transferred to nitrocellulose
membrane (BioRad). Immunoblotting was performed
with mouse monoclonal antibodies against histone H1,
b-tubulin and b-actin (Abcam, Cambridge, MA), or rabbit
polyclonal antibody against HIV-1 Gag (26). Secondary
antibodies were from Amersham (mouse) or Santa Cruz
(rabbit). Visualization was performed with Luminol
reagent (Santa Cruz Biotechnology, Santa Cruz, CA).
For siRNA transfection, COS cells were seeded at
5 104 cells per well in 35-mm plates. After 24 h, cells were
incubated with a mixture of Oligofectamine (Invitrogen)
and 10 ul of 20 mM siRNAs (Dharmacon) targeted to two
regions of RHA (5 ul of each) or scrambled sequences (29)
in OptiMEM media (Invitrogen) without serum. FBS
(Invitrogen) was added 4 h post-treatment and cells
were incubated at 378C in 5% CO2. After 72 h, siRNA
treatment was repeated with either 5 ml of scrambled
siRNAs or 5 ml of the RHA siRNAs. Cells were then
transfected with PCE HIV-gag reporter plasmids or
HTLV-1 proviral clones and pGL3 luciferase plasmid
using FuGene6, as described above.
RNA isolation and analysis
For in vitro transcription, linearized bicistronic reporter
plasmids that contain the T7 promoter upstream of the
reporter gene were incubated with MAXIscript T7
polymerase and cap analog (Ambion), followed by
treatment with poly(A) polymerase (Ambion), and sub-
sequent DNase treated (52). After addition of 0.5M
EDTA and incubation at 708C for 10min, the RNA was
isolated by acid phenol extraction and ethanol precipita-
tion. Cellular RNAs were isolated in Trizol following the
manufacturer’s protocol (Invitrogen). Total cellular RNA
and RNA extracts of nucleoplasm and cytoplasm were
subjected to two DNase treatments, extraction with acid
phenol, precipitation with ethanol and resuspension in
diethyl pyrocarbonate-treated water. For northern blot
analysis, 5 mg total RNA or 1 mg synthetic RNA was
separated on 1.2% agarose gels containing 5% formalde-
hyde, transferred to Duralon-UV membrane (Stratagene,
La Jolla, CA), and incubated with firefly luciferase (F-luc)
DNA probe. The probe was an F-luc restriction product
that had been gel purified and labeled with [a-32P]dCTP
by a random-primer DNA-labeling system (Invitrogen,
CA). The hybridization products were scanned with
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
For reverse transcription, polyAþmRNA was isolated
using Oligotex (Qiagen) and the mRNA was reacted with
Sensiscript reverse transcriptase (Qiagen) and random
hexamer primer for 1 h at 378C. Ten percent of the cDNA
preparation was used for real-time PCR with primers
complementary to HIV gag, b-actin, or gapdh and
Quantitect SYBR Green PCR (Qiagen, Germany) in a
Lightcycler (Roche, Germany). Standard curves to deter-
mine mRNA copy numbers were prepared with control
reactions with pHIVNL4-3, b-actin or gapdh plasmid in the
range of 102–108 copies. RNA co-immunoprecipitation
assays were performed as previously described (29).
Ribosomal profiles were prepared as described previously
(31). The samples were precipitated in warm ethanol and
RNA was extracted in Trizol as described above. cDNA
was created using Omniscript RT kit (Qiagen) and used
as a template for real-time PCR as described above.
RESULTS
SNV 50 UTR does not confer internal initiation
To assess IRES activity in selected retroviral 50 UTRs,
we evaluated the activity of bicistronic reporter plasmids
in transiently transfected 293 cells. The bicistronic
reporter transcript contains renilla luciferase (R-luc) in
the first cistron, which serves as the cap-dependent
translation reporter, and firefly luciferase (F-luc) in the
second cistron, which is used to measure IRES activity
(Figure 1A). The intercistronic region of pNoIRESfs
introduces three stop codons into the R-luc open reading
frame followed by a frameshift mutation (31). pNoIRESfs
was used to measure the baseline level of read-through
translation from R-luc into F-luc open reading frame.
pPolio contains the poliovirus IRES and was used to
measure efficient internal initiation of F-luc translation.
The pSNV derivatives contain the indicated SNV 50 UTR
sequence to evaluate the possibility that they support
internal initiation. Five independent transient transfection
assays were performed and the results of dual luciferase
assays were subjected to statistical analysis. A linear
model using R-Luc as a covariate was used to determine
whether or not F-Luc activity from pPolio was signifi-
cantly different from the other groups. Box plot analysis,
which summarized the mean and variance between the
Nucleic Acids Research, 2007, Vol. 35, No. 8 2631

samples, determined that F-Luc activity of pPolio and
pSNV/1-591 segregated in one group, whereas pSNV/260-
591 and pSNV/393-591 segregated with the negative
control pNoIRESfs (Figure 1A). The results of the five
replicate assays show that F-Luc activity from pPolio and
pSNV/1-591 is at least two logs greater than pNoIRESfs,
pSNV/260-591 or pSNV/393-591 (Figure 1A and B).
Northern blot analysis with 32P-labeled DNA probe
complementary to F-luc was used to evaluate the structure
of the bicistronic transcripts. Analysis of total RNA from
the transfected cells verified that pNoIRESfs, pPolio,
pSNV/260-591 and pSNV/393-591 expressed the expected
homogeneous 3.6 kb transcript population (Figure 1A).
The results indicated that IRES activity was not
conveyed by the distal subsections of the SNV 50 UTR.
By contrast, pSNV/1-591 expressed a heterogeneous
transcript population consisting of the expected 3.6 kb
transcript plus another prominent transcript of 2.5 kb.
Therefore, the observation of significant F-Luc activity
from pSNV/1-591 was associated with an aberrant F-luc
transcript. We concluded that the observed F-Luc activity
may be attributable to cap-dependent initiation of an
internally initiated or spliced F-luc transcript. Therefore,
we utilized an alternative approach to evaluate whether or
not the SNV 50 UTR supports cap-independent transla-
tion initiation.
As an alternative approach, we used infection with the
picornavirus EMCV to downregulate cap-dependent
translation and evaluated the effect on an SNV molecular
clone. D17 cells, which are permissive for SNV replication,
were permanently transfected with either SNV provirus,
pSNVgagluc, cap-independent F-Luc bicistronic reporter
CMV R-luc F-luc
pPolio
pNoIRESfs
pSNV/1-591
pSNV/260-591
pSNV/393-591
Polio
1–591
260–591
Three stop
codons + fs
393–591
~3.6 kb
1110987654
11
F-
Lu
c
(na
tur
al
log
tr
an
sfo
rm
ed
)
F-Luc (natural log transformed)
10
9
8
7
pNoIRESfs
pPolio
pSNV/260–591
pSNV/393–591
pSNV/1–591
6
5
4
6050403020100
*
A
B
R-Luc (RLU × 103)
Figure 1. SNV 50 UTR sequences do not confer IRES activity in bicistronic reporter plasmids. (A) Diagram of CMV IE-driven bicistronic reporter
gene that encodes renilla luciferase (R-luc) in the first cistron, firefly luciferase (F-luc) in the second cistron and indicated intercistronic regions. The
intergenic region of pNoIRESfs introduces three in-frame stop codons and a downstream frameshift mutation into the R-luc open reading frame for
terminating cap-dependent translation of R-luc. pPolio contains the poliovirus internal ribosome entry site. The pSNV plasmids contain SNV 50
UTR sequences with number relative to position in SNV RNA. Representative northern blot with F-luc DNA probe verifying expression of the
expected 3.6 kb transcript and an aberrant transcript marked with an asterisk. Five independent transfection assays were performed in 293 cells and
total cellular proteins were harvested at 48 h post-transfection and subjected to dual Luciferase assay. Box plot analysis summarizes the range of
F-Luc activity with internal line indicating mean and extending lines connecting extreme values. (B) Scatterplot analysis of the five replicate
experiments demonstrated that pPolio and pSNV/1-591 segregated separately from the other plasmids. Natural log transformation was used to adjust
non-normality and unequal variances.
2632 Nucleic Acids Research, 2007, Vol. 35, No. 8

plasmid pEMCV (31), or cap-dependent F-Luc expression
plasmid pGL3 (Promega) (Figure 2A). pSNVgagluc is
derived from pPB101 (44) and contains an in-frame fusion
of gag-pol with F-luc (Figure 2A). The D17 cell lines were
infected with EMCV (MOI40.5) and F-luc activity was
quantified at sequential time points. By 4 h post-infection,
cap-independent translation of F-luc from pEMCV
had increased by 200% (Figure 2B). In contrast,
F-Luc activity from the pGL3 cap-dependent control
failed to increase over time. The F-Luc production from
pSNVgagluc segregated with pGL3 and failed to increase
over time. These results indicate that SNV does not utilize
cap-independent internal initiation as a major mode of
translation initiation and is reliant on cap-dependent
initiation.
50 UTR of REV-A and HTLV-1 do not support
internal translation initiation
The bicistronic luciferase reporter plasmids were used to
evaluate the 50 UTR sequences of the avian lymphotropic
retrovirus, REV-A, which exhibits 90% sequence homol-
ogy with SNV, and HTLV-1, a genetically more complex
retrovirus. Four independent transient transfection assays
were performed in HeLa cells with pREV-A, pHTLV-1
and the control plasmids pPolio and pNoIRESfs. Results
of dual Luc assays showed that pREV-A and pHTLV-1
segregated with pPolio, similar to the results with pSNV/
1-580 in Figure 1 (Figure 3A and B). The results of
northern analysis of pREV-A and pHTLV-1 were also
reminiscent of the results of pSNV/1-591; a heterogeneous
transcript population was detected of the expected
3.6 kb transcript, plus smaller prominent RNA species
(Figure 3A). Therefore, the F-Luc activity of pREV-A and
pHTLV-1 is associated with the presence of aberrant
transcripts. Prominent aberrant transcripts were also
identified when the experiment was conducted in D17
cells (Figure 3C). We concluded that plasmid transfection
was not an appropriate assay for measuring IRES activity
in these particular sequences.
A documented approach to avoid false-positive IRES
activity associated with aberrant transcripts is direct
transfection of in vitro transcribed bicistronic RNAs
(53). To evaluate this method in our system, we generated
capped and polyadenylated bicistronic in vitro transcripts
and assessed internal initiation from pREV-A and
pHTLV-1 RNA by RNA transfection into HeLa cells.
Northern blot analysis verified expression of the expected
3.6 kb homogeneous transcript population and aberrant
transcripts were not detected (Figure 4). Five independent
RNA transfection experiments were performed and
results of dual Luc assays were used for statistical analysis.
Tukey pairwise comparisons showed that the mean F-Luc
activity of the pPolio positive control was significantly
higher then all other groups (P-value50.0001) (Figure 4).
In contrast, the mean F-Luc activity of pREV-A
and pHTLV were not statistically different from the
pNoIRESfs negative control (P-value 0.38 and 0.17,
respectively). The results of the RNA transfection assays
indicated that these 50 UTR sequences do not support
internal translation initiation from a bicistronic reporter
in HeLa cells. The lack of internal initiation prompted us
to examine the possibility that the 50 UTR of REV-A and
HTLV-1 contain a PCE to facilitate efficient translation.
50 LTR of divergent lymphotropic retroviruses
exhibit PCE activity
The hybrid SNV PCE-HIV gag reporter system was used
previously to identify PCE activity within the 50 proximal
UTR sequences of SNV, MPMV and cellular junD
(26,28,29). To test REV-A and HTLV-1 for PCE activity,
we constructed derivatives of the hybrid SNV-HIV gag
reporter plasmid (pYW100) that replaced the SNV LTR
with the LTR of REV-A or HTLV-1 (Figure 5).
Additional derivatives were constructed to evaluate the
LTRs of feline leukemia virus (FeLV), HTLV-2, bovine
leukemia virus (BLV) and equine infectious anemia virus
(EIAV) for PCE activity. The plasmids were transiently
transfected into 293 cells with Luciferase expression
plasmid (pGL3), and F-Luc activity was used to
standardize minor differences in transfection efficiency.
In experiments to test the LTRs of the genetically more
complex retroviruses, the gag reporter plasmids were
co-transfected with expression plasmid of the
F-luc envgagU5 RU3 U5RU3
R-luc EMCV F-luc
F-luc
pEMCV
pSNVgagluc
pGL3SV40
CMV
A
B
F-
Lu
c
(R
LU
×
10
3 )
10
20
30
40
50
60
0 1 3 4 652
pEMCV
pGL3
pSNVgagluc
Hours post-EMCV infection
Figure 2. SNV initiates translation in a cap-dependent manner.
(A) Diagram of SNV reporter provirus that contains F-luc in place
of pol, and control cap-independent F-luc reporter plasmid that
contains the encephalomyocarditis virus internal ribosome entry
site (pEMCV), and cap-dependent F-luc reporter plasmid, pGL3.
Transcription is driven by SNV U3, cytomegalovirus (CMV) or simian
virus 40 (SV40) promoter, respectively. (B) Total cellular proteins were
harvested at four times after infection with EMCV and equivalent
protein samples were subjected to F-Luc assay. Duplicate experiments
were performed and average Luciferase activity is presented. Relative
light units, RLU.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2633

corresponding transcriptional transactivator (HTLV-1,
HTLV-2 or BLV Tax and EIAV Tat), which is necessary
to trans-activate the viral promoter. Total cellular protein
was harvested 48 h post-transfection and Gag production
was measured by HIV Gag ELISA. Representative results
of three independent assays show that the positive control
SNV LTR (pYW100) facilitated Rev/RRE-independent
Gag production (Figure 5). PCE activity was also
observed in response to the LTRs of REV-A (pREV-
A100) and to a lesser extent, the gammaretrovirus FeLV,
and the deltaretrovirus HTLV-1. In response to the BLV
LTR, Gag production was near the detection limit of the
assay, while the HTLV-2 and EIAV reporters did not
produce detectable levels of Gag. To investigate PCE
activity conferred by the REV-A LTR, the 50 proximal
160-nt 50 UTR of pREV-A100, which corresponds to RU5
region of the LTR, was deleted in pREV-A205
(see Figure 6A). Comparison of pREV-A100 and pREV-
A205 determined that deletion of REV-A RU5 reduced
Rev/RRE-independent Gag production (Table 1). Gag
immunoblot verified the differences in Gag protein level
(Figure 6B). These results indicate the REV-A RU5 is
necessary for robust PCE activity.
To assess potential differences in steady-state gag RNA,
total cellular RNA was subjected to RT-PCR analysis.
After reverse transcription with random hexamer primer,
the cDNAs were subjected to PCR with primers com-
plementary to R and gag to amplify the region spanning
the 50 splice site. Electrophoresis of the RT-PCR products
verified expression by pREV-A100 and pYW100 of the
expected 520-bp product without aberrant gag RNAs
(Figure 6C). Control reactions without RT demonstrated
a lack of DNA contamination and control reactions on
the corresponding plasmid template demonstrated the
CMV R-luc F-luc
pPolio
pNoIRESfs
pREV-A
pHTLV-1
pPoliopNoIRESfs pREV-ApHTLV-1
Polio
1–580
1–263
Three stop
codons + fs
11109876
11
F-
Lu
c
(na
tur
al
log
tr
an
sfo
rm
ed
)
F-Luc (natural log transformed)
10
9
8
7
pNoIRESfs
pPolio
pREV-A
pHTLV-1
6
60 7050403020100
*
*
*
*
*
*
*
*
**
A
B C
R-Luc (RLU × 103)
Figure 3. Transfection of REV-A and HTLV-1 bicistronic reporters yield heterogeneous transcript population. (A) Diagram of CMV IE-driven
bicistronic reporter gene that encodes renilla luciferase (R-luc) in the first cistron, firefly luciferase (F-luc) in the second cistron and indicated
intercistronic regions. Representative northern blot with F-luc DNA probe verifying expression of the expected 3.6 kb transcript and aberrant
transcripts marked with an asterisk. Four independent transfection assays were performed in HeLa cells and total cellular proteins were harvested at
48 h post-transfection and subjected to dual Luciferase assay. Box plot analysis summarizes the range of F-Luc activity with internal line indicating
mean and extending lines connecting extreme values. (B) Scatterplot analysis of the four replicate experiments demonstrated that pPolio and pREV-
A and pHTLV-1 segregated separately from the pNoIRESfs plasmid. Natural log transformation was used to adjust non-normality and unequal
variances. (C) Northern blot with F-luc DNA probe indicates aberrant transcripts are also present in transfected D17 cells.
2634 Nucleic Acids Research, 2007, Vol. 35, No. 8

amplicon size. Real-time PCR analysis of poly(A)þ
mRNA was used to quantify steady-state gag RNA
among the transfectants. Comparison of pREV-A100
and pREV-A205 in two independent experiments deter-
mined that the presence of RU5 did not correlate with
increased steady-state gag RNA (Table 1). The results
indicated that the greater Gag protein production from
pREV-A100 compared to pREV-A205 is not attributable
to greater steady-state RNA. Comparison of the ratio of
Gag protein production relative to gag RNA from the
same transfectant determined that gag mRNA transla-
tional efficiency was 3–7-fold greater for pREV-A100
compared to pREV-A205 (Table 1).
Comparison between pREV-A100 and the SNV control
plasmid pYW100 indicated that pYW100 consistently
yielded greater abundance of gag mRNA and Rev/RRE-
independent Gag protein production (Table 1). To
determine if the difference is attributable to the activity
of the SNV U3 promoter region, we inserted SNV U3 in
place of REV-A U3 in pREV-A100 and created pCG211.
Results of three independent experiments determined
that replacement of REV-A U3 with SNV U3 did not
increase gag steady-state mRNA (compare pREV-A100
with pCG211) (Table 1). The copy number of steady-
state gag RNA from pCG211 is less than the SNV
U3-containing plasmids (pYW100 and pYW205).
Comparison of Gag production from pCG211 and
pYW205 verified that REV-A RU5 was sufficient to
increase Gag protein production. The ratio of Gag protein
level per gag mRNA was 5-fold greater for pCG211 than
pYW205. To address whether or not this activity is
specific for REV-A RU5, REV-A RU5 was replaced
1–263
Polio
Three stop
codons + fs
5 6 7 8 9 10 11
1–580
pHTLV-1
pPolio
pNoIRESfs
pREV-A
F-Luc (natural log transformed)
7mG p(A)R-luc F-luc
~3.6 kb
P=0.17
P=0.38
*P <0.0001
Figure 4. RNA transfection assays indicate that HTLV-1 and REV-A 50 UTR sequences lack IRES activity. Diagram of the synthetic bicistronic
transcripts that were transcribed in vitro, capped, polyadenylated and transfected into HeLa cells. Labels are as described in Figure 1. Northern blot
with F-luc probe verified the homogeneous transcript populations. Dual Luciferase assays were performed on total cellular protein at 20 h post-
transfection. Box plot analysis of the five independent experiments summarizes range of F-Luc activity with mean indicated by the internal line and
extending lines connect box to extreme values. Tukey method was used to measure the significance level of each group relative to pNoIRESfs.
Asterisk indicates P-value that is statistically different from pNoIRESfs group.
HIV gag-polU5 RU3
Selected LTR
SNV
REV-A
FeLV
HTLV-1
BLV
HTLV-2
EIAV
Selected
LTR
Gag protein
(pg/ml)
Measurement of Rev/RRE-
independent Gag production
100037
40157
6332
2851
734
<MD
<MD
Figure 5. Survey for PCE activity in the long terminal repeat (LTR)
of seven divergent retroviruses. Diagram of PCE-HIVgag reporter
plasmid used to assay the LTR of selected retroviruses for PCE activity.
Observation of Rev/Rev responsive element (RRE) independent
Gag protein production from unspliced HIV gag reporter RNA
indicates PCE activity. The results are representative of three
independent transfection assays in 293 cells. Gag in cellular protein
was quantified by ELISA.5MD, below the detection limit of the assay
(15 pg/ml).
Nucleic Acids Research, 2007, Vol. 35, No. 8 2635

with MLV RU5 (pSH603). Gag protein production was
below the minimum detection level of the assay, indicating
that PCE activity is conferred by REV-A RU5, but not
MLV RU5.
To investigate a possible effect of REV-A RU5 on the
cytoplasmic accumulation of gag mRNA, the nucleoplasm
and cytoplasm were fractionated, and polyadenylated
nuclear and cytoplasmic mRNAs were isolated, reverse
transcribed and quantified by real-time PCR. Western
blot analysis of the fractionated proteins with antisera
to nuclear protein histone H1 and cytoplasmic protein
b-tubulin verified effective sub-cellular fractionation of the
nucleoplasm and cytoplasm (Figure 7A). Comparison of
gag mRNA copy numbers from pREV-A100 and pREV-
A205 determined minor differences in gag mRNA level in
the nucleus or cytoplasm (Figure 7B). The percentage of
gag mRNA in the cytoplasm was not increased by REV-A
RU5 (compare pREV-A100 and pREV-A205), similar to
results with SNV PCE (compare pYW100 and pYW205)
(26,28). The ratios of Gag protein level relative to gag
mRNA copy number demonstrated that the presence of
REV-A RU5 or SNV RU5 correlated with an increased
translational efficiency of cytoplasmic gag mRNA
(Figure 7B). We concluded that REV-A 50 RU5 contains
a PCE that facilitates the translational utilization of
cytoplasmic gag RNA.
RHA is necessary for REV-A PCE activity and gag
expression fromHTLV-1 provirus
To determine if RHA is necessary for REV-A PCE
activity, siRNAs were used to downregulate endogenous
RHA. COS cells were transfected with siRNAs directed
against RHA (DHX9) or the negative control scrambled
sequence (Sc siRNA) (29). This siRNA treatment regi-
ment was previously shown to specifically downregulate
RHA without reducing cell viability, the rate of global
cellular RNA and protein synthesis or gene-specific
translation of gapdh and F-luc (29). Seventy-two hours
after siRNA transfection, the cells were transfected
with siRNAs and either pREV-A100 or pYW100
BA
C
pR
EV
-A
10
0
pR
EV
-A
10
0
pREV-A100
pR
EV
-A
20
5
pY
W1
00
pY
W2
05
pY
W1
00pYW100
RT:
bp
1000
850
650
500
400
300
200
100
+ _ + _
pC
G2
11
Mo
ck
Gapdh
GagPr55U3 R U5 HIV gag-pol
U3 HIV gag-pol
U3 R U5 HIV gag-pol
U3 HIV gag-pol
U3 R U5 HIV gag-pol
pYW100
pYW205
pREV-A100
pREV-A205
pCG211
Figure 6. REV-A RU5 facilitates expression of HIV gag reporter RNA. (A) Diagram of PCE-gag reporter constructs. Gray rectangles, designated
U3, R, U5 segments of SNV LTR; black rectangles, designated U3, R, U5 segments of REV-A LTR, white rectangle, HIV gag open reading frame
within HIV intron. (B) Results of representative transfection assays in 293 cells with indicated PCE reporter plasmids. Immunoblot analysis of total
cellular protein with antiserum to HIV-1 Gag or Gapdh. (C) RT-PCR analysis of RNA with PCR primers complementary to R (15–37) and gag
(116 bp distal to gag ATG)-verified expression of the unspliced gag RNA. þ/
indicates presence or absence, respectively of reverse transcriptase
(RT). Indicated plasmid was used as a template in control reactions and indicates the expected size of each RNA amplicon. Results of Gag ELISA
and RNA quantification are summarized in Table 1.
Table 1. gag RNA and Gag protein production from PCE reporter
plasmids
Gaga
Replicate PCE reporter
plasmid
Protein
(pg/ml)
RNA
(103)
Protein:RNA
(relative to U3 only)
1 pREV-A100 53 877 4.879 2.9
pREV-A205 26 425 6.890 1.0
pYW100 1 14 040 10.239 2.8
pYW205 29 093 7.193 1.0
pCG211 54 903 2.888 4.8
2 pREV-A100 1 00 114 1.922 7.1
pREV-A205 48 842 6.720 1.0
pYW100 2 36 921 6.298 2.3
pYW205 61 721 3.707 1.0
pCG211 1 39 966 1.813 4.7
aCultures of 293 cells were transfected with indicated reporter plasmids
and total cellular protein and RNA were isolated. Gag protein
production was determined by ELISA. The minimum detection limit
of the assay was 15 pg/ml. RNA was subjected to reverse transcription
and real-time PCR with gag and b-actin primers. Values represent gag
copy number per nanogram standardized to b-actin copy number.
2636 Nucleic Acids Research, 2007, Vol. 35, No. 8

gag RNA was changed, and a minority of gag RNA is
associated with polyribosomes (12%). By comparison,
the majority of gapdh RNA remained associated with
polyribosomes (65 and 50%, respectively). Effective
downregulation of RHA was verified by immunoblot
(Figure 9C). These results demonstrate that RHA is
necessary for efficient HTLV-1 gag translation. We
conclude that RHA is necessary for efficient Gag
production from REV-A PCE reporter plasmid and
from HTLV-1 provirus.
RHA is necessary for PCE enhancement of Gag production
fromHTLV-1 Rex-deficient provirus
The observations that SNV PCE requires interaction with
RHA to facilitate HIV-1 Rev/RRE-independent Gag
production (29) and that the SNV LTR facilitates Rex/
RxRE-independent expression of BLV structural gene
vectors (47,54) compelled us to examine whether or not
SNV PCE can facilitate Rex/RxRE-independent expres-
sion of the related deltaretrovirus HTLV-1. To address
this issue, we constructed chimeric HTLV-1 proviruses
with an insertion of SNV PCE between the U3 and
RU5 regions of the 50 LTR (Figure 10A). The chimeric
proviruses encode either wild-type Rex (wtHTLV,
wt/PCE) or are Rex-deficient by introduction of a stop
codon (HTLVRex-, Rex-/PCE) (Figure 10A) (50).
The parental and chimeric proviruses were transfected
into 293 cells, and total cellular protein and RNA were
isolated. Gag protein production was quantified by
ELISA, gag mRNA was reverse transcribed and quanti-
fied by real-time PCR. Results of two replicate experi-
ments determined that insertion of SNV PCE upstream of
HTLV-1 RU5 did not disrupt HTLV Gag production
(compare wtHTLV-1 and wt/PCE) (Table 2). As expected,
cells transfected with HTLVRex- yielded Gag levels near
or below the limit of detection, and the steady-state
gag RNA level was consistently lower from the proviruses
gag 2303
191
3903
479
353
59
412
4
187
42
662
344
651
16
334
1gapdh
87654321RNA
85gapdh 113
1202
100
39
56
381
208
7142
31
325
3
202
2
325gag
87654321RNA
RHA
Sc RHA
Actin
65%
75%
RNA on
polysomes
Polysomes
Polysomes
50%
12%
RNA on
polysomes
Sc siRNA
A 2
54
A
25
4
RHA siRNA
A
B
C
Figure 9. RHA promotes translation of HTLV-1 gag mRNA. COS cells were transfected with siRNAs that target two regions in RHA or scrambled
sequence (Sc), incubated for 48 h, transfected with HTLV-1 provirus and incubated for 48 h. Cytoplasmic extracts were separated on 15–50% linear
sucrose density gradients. Ribosomal RNA profiles (A254) of cells treated with (A) Sc siRNAs or (B) RHA siRNAs. RNA was extracted from each
gradient fraction, reverse transcribed and subjected to real-time PCR to quantify HTLV-1 gag RNA copies. Charts below each profiles indicate 102
copies of gag or gapdh present in equivalent fractions. (C) Immunoblotting with antisera against RHA and b-actin verifies efficient downregulation of
RHA and indicates equivalent sample loading, respectively.
2638 Nucleic Acids Research, 2007, Vol. 35, No. 8

that lack Rex (50). Insertion of SNV PCE into the Rex-
deficient HTLV-1 provirus (Rex-/PCE) increased Gag
production by418-fold (compare HTLVRex- and Rex-/
PCE). The results indicate that SNV PCE has the capacity
to facilitate Rex/RxRE-independent Gag production
from a Rex/RxRE-deficient HTLV-1 provirus. The
ratios of Gag protein relative to gag mRNA indicated
that insertion of SNV PCE into Rex-deficient provirus
robustly increased translational utilization of the HTLV-1
gag transcripts (420-fold).
To assess the effect of RHA on Gag protein production
from the HTLV-1 proviruses, COS cells were transfected
with the RHA siRNAs, incubated for 72 h and RHA
downregulation was verified by RHA immunoblot on an
aliquot of the cells (data not shown). The treated cells
were transfected with siRNA, HTLV-1 provirus and
pGL3 and incubated for 48 h. RHA immunoblot indicated
sustained downregulation of RHA (Figure 10B). HTLV
Gag ELISA indicated that RHA downregulation signifi-
cantly decreased Gag production from each of the
proviruses (Figure 10B). RHA downregulation reduced
Gag production from wtHTLV-1 and wt/PCE to510%
and eliminated Gag production from Rex-/PCE provirus.
The results are in agreement with the ribosomal profile
analysis (Figure 9) and demonstrate that RHA is
necessary for efficient expression of HTLV-1 Gag struc-
tural protein. Furthermore, the results indicate that
SNV PCE facilitates Gag protein production from a
Rex/RxRE-deficient HTLV-1 provirus in an RHA-
dependent manner.
DISCUSSION
This study assessed the possibility that selected retroviral
50 UTRs confer PCE activity or IRES activity. We
surveyed seven retroviruses for PCE activity. Results of
PCE reporter assays determined that the 50 LTR of REV-
A, HTLV-1 and FeLV contain a 50 terminal PCE, which
can substitute for SNV PCE to facilitate Rev/RRE-
independent expression of HIV gag RNA. The analysis
further determined that the RU5 region of the REV-A
LTR is necessary for the Rev/RRE-independent Gag
production, and REV-A PCE activity is attributable to
increased translation efficiency of the REV-A-PCE gag
RNA, rather than increased cytoplasmic accumulation of
the gag RNA (29). This observation that PCE mutation
disrupts translational utilization of the mRNA without
detectable change in the cytoplasmic abundance supports
the model that PCE–RHA interaction overcomes seques-
tration of the transcript in RNA storage granules (29).
The experiments did not identify PCE activity in the 50
LTR of HTLV-2, EIAV or MLV in 293 cells. Possible
explanations are that these sequences do not contain a 50
terminal PCE or that the level of LTR-driven transcrip-
tion in 293 cells was insufficient for detection of Gag
4772ATGCCCAAG4780
4772ATGCCTTAG4780
1TGGATATCATGGATCCT17
U3 R U5 U3 R U5
gag
pol
pro env
tax
rex
orf-I
orf-II
wtHTLV-1
EcoRV BamH I
SNV-PCE
HTLVRex-
hbz
*
Gag (pg/ml):
Sc RHA Sc RHA Sc RHA Sc RHA
Gapdh
RHA
siRNA:
529 40 <MD<MD 469 26 <MD100
Provirus: Rex-/PCEwt/PCERex-wt
A
B
Figure 10. SNV PCE can enhance translation from HTLV-1 Rex-
deficient provirus. (A) Diagram of the HTLV-1ACH provirus. Terminal-
labeled rectangles depict U3, R, U5 regions of the long terminal
repeats. Labeled rectangles indicate gag, pro, pol, env, tax, rex, HBZ
open reading frames, orf I and orf II. The wild-type DNA sequence
near the Rex start codon (ATG) is labeled (wtHTLV-1). Bold letters
indicate the site-directed mutation to introduce a stop codon in the Rex
open reading frame (HTLVRex-). Location of SNV PCE insert is
shown with bold letters indicating site-directed mutations that
introduced the indicated restriction sites. Numbering refers to RNA
sequence. (B) COS cells were treated with scrambled (Sc) and RHA
siRNAs as described in Figure 8. Concurrent with second siRNA
transfection, the cells were co-transfected with indicated HTLV-1ACH
provirus. Forty-eight hours later, total cellular protein was harvested
and subjected to immunoblot with RHA antiserum to assess down-
regulation and Gapdh antiserum to control for sample loading. Results
of Gag ELISA are summarized below the immunoblot;5MD, less than
the minimum detection limit of the assay (25 pg/ml).
Table 2. Gag protein and RNA expression from HTLV-1 proviruses
Gag
Replicate HTLV-1
provirus
Gaga pg/ml gag RNAb Ratio of Gag
protein:RNA
1 wtHTLV-1 3125 417.5 7.5
HTLVRex 31 228.4 0.1
wt/PCE 4663 662.5 7.0
Rex-/PCE 576 267.3 2.1
2 wtHTLV-1 2352 392.7 6.0
HTLVRex 5MD 237.3 5MD
wt/PCE 4118 729.7 5.6
Rex-/PCE 796 285.4 2.8
aCultures of 293 cells were co-transfected with indicated provirus
and pGL3. Total cellular protein was isolated and Gag quantified
by ELISA; 5MD, below the minimum detection limit of the assay
(25 pg/ml).
bgag copies per nanogram total RNA standardized to gapdh as
determined by real-time PCR.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2639

protein production. Further analysis is necessary to assess
these possibilities.
Our findings show that the 50 UTR of REV-A HTLV-1
and SNV do not support IRES activity in bicistronic
RNA. Stringent bicistronic reporter assays by plasmid
transfection, RNA transfection of synthetic RNAs, and
RNA analysis identified a lack of IRES activity in the 50
UTR of REV-A, HTLV-1 and SNV. Our findings contrast
with published analysis of a bicistronic neomycin phos-
photransferase reporter vector that identified IRES
activity in REV-A after selection with G418 (32). IRES-
like activity has also been identified from HTLV-1R
expressed adjacent to the SV40 early gene leader (33,34).
Here, we identified that bicistronic reporter activity was
detectable in the REV-A and HTLV-1 UTR, but is
attributable to heterogeneous F-luc transcripts, rather
than authentic IRES activity. A second, complementary
approach assessed SNV translation initiation in the
natural context of an SNV provirus. Results using
EMCV to inhibit cap-dependent translation initiation
determined that SNV is reliant on a cap-dependent
initiation mechanism. Our results do not completely
exclude the possibility of internal initiation, but indicate
that cap-independent internal initiation is not a major
mode of translation initiation for SNV in asynchronous
cells. We speculate that retroviral 50 UTR is a pliable
template for mRNA translation initiation that is reorga-
nized by changes in nucleoprotein architecture. The
reorganization allows the virus to sustain translation
during oscillations in the translation capacity of the cell,
which alternatively favor cap-dependent initiation or cap-
independent internal initiation (8). We speculate that the
capacity to respond is orchestrated by alternative virus–
host interactions: PCE–RHA and IRES–host factor,
respectively.
The specific interaction between RHA and the SNV
PCE and the PCE at the 50 RNA terminus of the cellular
junD gene has been shown to be necessary for efficient
junD translation (29). Here, we verified that RHA is
essential for PCE activity conferred by REV-A PCE
reporter mRNA and for efficient translation of HTLV-1
gag mRNA from HTLV-1 provirus. Our results implicate
RHA as an important translation regulatory effector of
multiple lymphotropic retroviruses. Our bioinformatic
searches have not identified a common primary sequence
motif among SNV, MPMV, REV-A, HTLV-1, FeLV and
junD PCE. However, genetic and co-immunoprecipitation
experiments have identified that structural motifs in SNV
PCE are necessary for the selective recruitment of RHA
(29). We speculate that the growing collection of viral and
cellular PCEs share related secondary or tertiary struc-
tural motifs that convey selective interaction with RHA.
HTLV-1 is dependent on the virally encoded Rex
protein in association with the cis-acting RxRE for
efficient export of viral transcripts. Here, we have shown
that the PCE of SNV, a genetically simple retrovirus, is
capable of facilitating Rex/RxRE-independent expression
of HTLV-1 provirus. This finding is an example of the
interchangeability of viral elements to facilitate robust
gene expression in diverse gene expression systems. SNV
PCE has also been shown to enhance translational
efficiency of lentiviral vector transgene (55), modulate
expression of BLV structural gene vectors (47,54) and to
synergize with a heterologous transcriptional enhancer
and constitutive transport element to increase protein
yield (51,55).
Downregulation of endogenous RHA decreased Gag
production from both wtHTLV-1 and PCE-containing
Rex-deficient HTLV-1, implying that RHA is necessary
for efficient translation of HTLV-1 transcripts that
accumulate in the cytoplasm in either a Rex-dependent
or Rex-independent manner. Not unexpectedly, the
insertion of SNV PCE did not completely restore Gag
production to the level of wtHTLV-1. This is attributable
to impaired cytoplasmic accumulation of HTLV-1 tran-
scripts because of the lack of Rex. We expect that SNV
PCE facilitates Rex/RxRE-independent expression by
upregulating the translational utilization of residual
cytoplasmic gag mRNA that accumulates in the cyto-
plasm in the absence of Rex.
We propose that RHA associates with the 50 proximal
SNV PCE in PCE/Rex- and the PCE within HTLV-1 RU5
in wtPCE. This interaction facilitates RNP remodeling
that promotes ribosome scanning and efficient translation
initiation. A similar model is proposed for PCE/RHA
translational stimulation of junD mRNA template as
described in Hartman et. al, 2006 (29).
Recent studies with HIV-1 suggest that RHA is
important for the process of reverse transcription and
functions in an RNA-dependent manner to enhance
infectivity of viral particles (56). If this phenomenon is
also true for HTLV-1, then the role of RHA in HTLV-1
pathogenesis may be multifaceted. A potential mechanism
is that RHA is packaged into viral particles, possibly in
association with the HTLV-1 PCE and promotes RNA–
protein and/or RNA–RNA remodeling that facilitates
reverse transcription. After provirus formation and
transcription of nascent viral RNA, RHA would interact
with PCE to facilitate translation of the viral genome. The
facilitation of efficient translation of viral structural and
enzymatic proteins with the process of reverse transcrip-
tion would function synergistically to promote infectivity
and pathogenesis of HTLV-1. Compliant with this model,
we did not observe PCE activity in HTLV-2, which
correlates with reduced replication capacity and patho-
genesis of HTLV-2 in relation to HTLV-1 (46).
ACKNOWLEDGEMENTS
We thank Mr. Tim Vojt for expert figure preparation, Dr.
Andrew Dangel for plasmid construction, Dr. Wendy
Maury for EIAV plasmids, Dr. Lawrence Mathes for
FeLV plasmid and Mr. Parul Gulati for biostatistics
assistance. This work was supported by grants from the
National Institutes of Health (P01CA16058 and
P30CA100730). Funding to pay the Open Access publica-
tion charge was provided by OHIOlink.
Conflict of interest statement. None declared.
2640 Nucleic Acids Research, 2007, Vol. 35, No. 8

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