ral
ssBioMed Cent
Retrovirology
Open Acce
Research
Coordinate enhancement of transgene transcription and
translation in a lentiviral vector
Alper Yilmaz
1,5
, Soledad Fernandez
3,4
, Michael D Lairmore
1,2,4,5
and
Kathleen Boris-Lawrie*
1,2,4,5
Address:
1
Center for Retrovirus Research and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, 43210, USA,
2
Department of Molecular Virology, Immunology & Medical Genetics, The Ohio State University, Columbus, OH, 43210, USA,
3
Center for
Biostatistics, The Ohio State University, Columbus, OH, 43210, USA,
4
Comprehensive Cancer Center, The Ohio State University, Columbus, OH,
43210, USA and
5
Molecular, Cellular & Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, 43210, USA
Email: Alper Yilmaz - yilmaz.11@osu.edu; Soledad Fernandez - fernandez.71@osu.edu; Michael D Lairmore - lairmore.1@osu.edu;
Kathleen Boris-Lawrie* - boris-lawrie.1@osu.edu
* Corresponding author
Abstract
Background: Coordinate enhancement of transgene transcription and translation would be a potent approach
to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many
vector transgenes are complementary DNA (cDNA). The lack of splicing can significantly reduce the efficiency of
their translation. Some retroviruses contain a 5' terminal post-transcriptional control element (PCE) that
facilitates translation of unspliced mRNA. Here we evaluated the potential for spleen necrosis virus PCE to
stimulate protein production from HIV-1 based lentiviral vector by: 1) improving translation of the internal
transgene transcript; and 2) functionally synergizing with a transcriptional enhancer to achieve coordinate
increases in RNA synthesis and translation.
Results: Derivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and
cytomegalovirus immediate early enhancer (CMV IE). Results from transfected cells and four different transduced
cell types indicate that: 1) PCE enhanced transgene protein synthesis; 2) transcription from the internal promoter
is enhanced by CMV IE; 3) PCE and CMV IE functioned synergistically to significantly increase transgene protein
yield; 4) the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5)
differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6) the
lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in
transduced cells.
Conclusion: PCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene.
Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE
transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of
the vector provirus into chromatin correlated with reduced cytoplasmic accumulation of PCE transgene RNA.
We speculate that epigenetic modulation of promoter activity altered cotranscriptional recruitment of RNA
processing factors and reduced the availability of fully processed transcript or the efficiency of export from the
nucleus. Our results provide an example of the dynamic interplay between the transcription and post-
transcription steps of gene expression and document that introduction of heterologous gene expression signals
Published: 15 February 2006
Retrovirology 2006, 3:13 doi:10.1186/1742-4690-3-13
Received: 06 January 2006
Accepted: 15 February 2006
This article is available from: http://www.retrovirology.com/content/3/1/13
© 2006 Yilmaz et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10
(page number not for citation purposes)
can yield disparate effects in transfected versus transduced cells.

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
Background
A challenge inherent to many gene delivery systems is effi-
cient expression of the vector transgene. Enhancement of
transcription has been a thoroughly investigated target to
improve vector gene expression. For example, introduc-
tion of a constitutive viral transcription enhancer or a tis-
sue-specific cellular promoter has been utilized widely to
stimulate synthesis of vector transgene RNA [1-4]. In addi-
tion to high level synthesis of RNA, efficient post-tran-
scriptional expression is a potent target to improve vector
gene expression by maximizing the protein yield per mol-
ecule of transgene transcript. Notably, many vector trans-
genes are complementary DNA (cDNA) copies of the
natural intron-containing gene. The elimination of
introns is an advantageous approach for reducing of the
size of the vector transcript to conform to the packaging
capacity of the vector virus. This approach is advantageous
in vectors with limited packaging size, as is the case for ret-
roviral vectors [5,6]. However the elimination of intronic
sequences can significantly reduce protein yield because
the process of splicing promotes the translation of intron-
containing genes [7-10]. This activity is attributed to a
multiprotein complex that is deposited at exon junctions
as a consequence of splicing [11,12]. The elimination of
intronic sequences can reduce protein yield in a range of a
factor of 2 to 30 [13,14]. Therefore the elimination of
introns from a transgene may reduce protein yield per
molecule of transgene transcript.
Recently, a unique 5' terminal positive posttranscriptional
control element (PCE) was identified in the 5' long termi-
nal repeat (LTR) of two simple retroviruses, spleen necro-
sis virus (SNV) and Mason-Pfizer monkey virus (MPMV)
[15,16]. PCE stimulates translation of non-spliced RNA
[16,17]. SNV PCE is a compact 165 nt orientation-
dependent RNA element that is composed of two func-
tionally redundant stem-loop structures that present
unpaired nucleotides for interaction with the ubiquitous
host protein RNA helicase A [[18], T. Hartman and K.
Boris-Lawrie, manuscript submitted]. PCE is not strictly
position-dependent and sustains activity when reposi-
tioned to at least 300 nt downstream of the transcription
start site [17]. In addition, PCE facilitates expression of
unspliced gag-pol RNA of HIV-1 and the parental retrovi-
rus, SNV [[15], T. Hartman, S. Hull and K. Boris-Lawrie,
unpublished].
Results from experiments with cDNA expression plasmids
determined that PCE stimulates protein yield from non-
spliced mRNA by 7 to 10-fold [17]. Quantitative RNA
analysis showed that the increased protein production
was not attributable to modulation of steady state RNA
level or nuclear export. Rather, the increased protein pro-
function as an internal ribosome entry site to stimulate
internal initiation on bicistronic reporter RNA [17]. These
findings and the determination that PCE requires nuclear
interactions for stimulation of translation [19] indicates
that PCE is a novel 5' terminal cap-dependent translation
enhancer of nonspliced RNA.
In addition to its functional activity, other properties
make PCE an excellent candidate for improving transla-
tional efficiency of vector transgene mRNA. First, PCE
functions in a wide variety of cells in concert with ubiqui-
tously expressed host effector protein. Second, PCE stim-
ulates translation of non-spliced mRNA template, which
is a common form of vector transgene mRNA. Third, PCE
exhibits flexibility in position relative to the transcription
start site, which provides versatility during vector con-
struction. The first goal of this study was to test the
hypothesis that SNV PCE increases the translational effi-
ciency of lentiviral vector transgene mRNA. In addition,
we reasoned that coordinate enhancement of transgene
Genomic structure of self-inactivating lentiviral vectors that lack r contain PCE translation enhanc rFigure 1
Genomic structure of self-inactivating lentiviral vec-
tors that lack or contain PCE translation enhancer.
HIV-1 based lentiviral vectors were derived from pHR' [36].
Black rectangles represent HIV-1 long terminal repeats; PBS,
primer binding site; Ψ, the core packaging signal; extended
packaging signal that corresponds to 350 nt HIV-1 gag open
reading frame; 5'ss and 3'ss, splice sites; RRE, Rev responsive
element; PPT, polypurine tract; ∆ indicates deletion of HIV-1
promoter sequences between -418 to -18; white boxes rep-
resent SNV sequences; Prom corresponds to spleen necrosis
virus (SNV) U3 promoter; PCE is the 165 nt RU5 region of
SNV; CMV IE, cytomegalovirus immediate early enhancer.
U3-Luc
PCE-Luc
IE-U3-Luc
IE-PCE-Luc
9
RRE luc
PBS
5’ss 3’ss
5’ss 3’ss
5’ss 3’ss
5’ss 3’ss
extended
packaging signal
extended
packaging signal
extended
packaging signal
extended
packaging signal
ppt
$prom
9
luc
PBS
ppt
PCEprom $
9
luc
PBS ppt
$prom
CMV IE enhancer
CMV IE enhancer
9
RRE luc
PBS ppt
PCEprom $
RRE
RREPage 2 of 10
(page number not for citation purposes)
duction was due to increased ribosome association. Addi-
tional experimentation determined that PCE does not
transcription and translation has significant potential for
synergistically improving efficiency of transgene expres-

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
sion in lentiviral vector and in other gene expression sys-
tems. The promoter of the lymphotropic SNV is
constitutively active in a wide variety of cells types from
different species [15,20-22]. The promoter encodes two
46 and 23 base-pair repeats with strong enhancer activity
and does not require virus-encoded transcription factor to
regulate transcriptional efficiency [21]. We constructed a
series of vectors to test whether the combination of PCE
and a strong heterologous transcriptional enhancer yields
a synergistic increase in protein production. Quantitative
analysis of RNA and protein levels were used to character-
ize the effect of PCE on vector RNA in transfected and
transduced cells. The results indicate that PCE and
cytomegalovirus immediate early (CMV IE) transcription
enhancer function synergistically to significantly improve
transgene protein output.
Results
CMV IE and PCE function synergistically to increase
protein output in transfected cells
A series of HIV-1 based self-inactivating lentiviral vectors
were constructed that lack or contain PCE and CMV IE
(Figure 1). The vector luciferase (luc) transgene was
expressed from an internal transcription unit under the
control of the constitutive SNV promoter. The vectors lack
or contain SNV PCE and the CMV IE transcriptional
enhancer and are designated U3-Luc, PCE-Luc, and IE-U3-
Luc and IE-PCE-Luc, respectively.
The vectors were transfected into 293 cells and two days
post-transfection, total cellular protein was harvested for
Luc assay. Comparison of U3-Luc and PCE-Luc demon-
strated that PCE increased Luc activity by 4 to 7-fold
(Table 1). Introduction of CMV IE produced a 2.4- to 4.4-
fold increase in Luc production (compare U3-Luc with IE-
UE-Luc and PCE-Luc with IE-PCE-Luc). Comparison of
U3-Luc and IE-PCE-Luc indicated that the combination of
PCE and CMV IE function synergistically to increase gene
expression.
PCE increases the translational efficiency of lentiviral
vector RNA
Northern blot analysis of total cellular RNA was per-
formed to compare the levels of steady state transgene
mRNA. Three replicate Northern blot experiments were
performed with radiolabeled probe complementary to the
luc open reading frame or glyceraldehyde-3-phosphate
dehydrogenase (gapdh) to control for RNA loading. The
experiments demonstrated that the vectors express luc
transcript of the expected size and that PCE-Luc and U3-
Luc displayed similar levels of steady state RNA (Figure
2A). In this representative experiment, luc mRNA levels
from PCE-Luc and U3-Luc RNA were 2.2 × 10
5
phos-
phorimager units (PI) and 1.8 × 10
5
PI, respectively (Fig-
ure 2B). Introduction of CMV IE produced an equivalent
2-fold increase in luc RNA level in either the presence or
absence of PCE (IE-PCE-Luc, 5.0 × 10
5
PI and IE-U3-Luc,
3.6 × 10
5
PI) (Figure 2B). Comparison of the level of Luc
protein to luc RNA showed that addition of PCE corre-
lated with a 4-fold increase in Luc protein (Figure 2B).
Ribosomal profile analysis determined that ribosome
association was greater for the PCE-containing vector than
the PCE-lacking vector (data not shown). The results indi-
cate that combination of CMV IE and PCE yielded a syn-
ergistic increase in vector transgene expression in the
transfected cells.
CMV IE and PCE function synergistically to increase
protein yield in transduced cells
Next we sought to determine whether the coordinate
increases in vector transgene expression were sustained in
transduced cells. The vector viruses were propagated by
co-transfection of 293T cells with each vector, HIV-1
helper plasmid and VSV-G expression plasmid. ELISA was
Table 1: The combination of PCE and CMV IE increased Luc activity in transfected 293 cells
Luc activity (Relative Light Units)
a
Replicate Experiment
Vector 1234
U3-Luc 2,955 ± 171 (1)
b
3,809 ± 207 (1) 3,605 ± 3 (1) 3,796 ± 700 (1)
PCE-Luc 20,810 ± 559 (7.0)* 19,644 ± 343 (5.1)* 14,756 ± 382 (4.0)* 15,110 ± 842 (3.9)*
IE-U3-Luc 10,490 ± 159 (3.5) 13,174 ± 228 (3.4) 12,945 ± 2,677 (3.6) 16,676 ± 435 (4.4)
IE-PCE-Luc 49,870 ± 28 (16.8)* 48,085 ± 90 (12.6)* 39,485 ± 7,303 (11)* 50,424 ± 1,952 (13)*
a
Two-days post-transfection with the indicated vector, which encodes firefly Luciferase (Luc) and Renilla luciferase control plasmid, total cellular
protein was harvested and relative Luciferase levels were measured by chemiluminescence assay. Luc level was standardized to cotransfected
Renilla Luc and results are presented of four independent experiments performed in duplicate or triplicate. ANOVA with repeated measures
determined that increases in response to PCE and IE were significant as indicated by * (p-values of 0.0008 and < 0.0001, respectively).
b
(), Fold difference relative to U3-luc vector.Page 3 of 10
(page number not for citation purposes)
PCE and CMV IE produced a cumulative 11 to 17-fold
increase in protein production. The results indicate that
used to measure the capsid Gag levels and equal amounts
of Gag were used for transduction by spinoculation of

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
HeLa human fibroblast cells, CEM-A human T cells, D17
canine osteosarcoma cells, and 293 human embryonic
kidney cells. Forty-eight hours post-transduction, the
transduced cells were harvested and Luc activity in total
cellular protein was measured.
The PCE-containing vectors exhibited increased Luc pro-
duction in all four target cells (Table 2). The magnitude of
increase in response to PCE was 2 to 4-fold (Table 2, com-
pare U3-Luc and PCE-Luc). The magnitude of increase in
response to CMV IE was an additional 2-fold (Table 2,
compare U3-Luc with IE-U3-Luc and PCE-Luc with IE-
PCE-Luc). Comparison of U3-Luc and IE-PCE-Luc indi-
tion. These increases were lower in magnitude than the
increases observed in the transfected cells (Table 1). Real-
time PCR was performed to evaluate provirus copy
number and revealed similar levels of vector provirus in
transduced 293 cells. In this representative experiment,
the copy numbers for PCE-Luc, U3-luc, IE-PCE-Luc and
IE-U3-Luc were 4.24 × 10
3
; 6.31 × 10
3
; 3.83 × 10
3
; and
2.07 × 10
3
copies/ng, respectively. The results showed that
the transduction efficiency was similar between the vec-
tors and was not affected by introduction of PCE or CMV
IE.
Vector transduction correlates with reduced cytoplasmic
accumulation of PCE-Luc RNA
Northern blot assay was used to evaluate steady state luc
RNA levels in three replicate experiments. Northern blot
analysis of total cellular RNA determined that after trans-
duction, the PCE-containing vectors expressed less steady
state luc RNA compared to their PCE-lacking derivative
(compare U3 and PCE, IE-U3 and IE-PCE, Figure 3A). Fig-
ure 3B summarizes the luc RNA levels standardized to
gapdh loading control for this particular experiment. This
trend differed from the results in transfected cells, wherein
the steady state luc levels were not lower in the presence
of PCE (Figure 2). Introduction of CMV IE produced an
equivalent 2-fold increase in luc RNA level in either the
presence or absence of PCE, which was similar in magni-
tude to the increase in transfected cells (compare PCE and
IE-PCE, U3 and IE-U3, Figure 2). Two of the possible
explanations for the lower steady state luc RNA in
response to PCE are that PCE lowers the cytoplasmic accu-
mulation or the stability of the luc transcript.
Quantitative analysis of nuclear and cytoplasmic RNA lev-
els was used to investigate possible differences in cytoplas-
mic accumulation. The transduced cells were fractionated
into nucleoplasm and cytoplasm, RNA was harvested and
subjected to reverse transcription, and cDNA levels were
quantified by real time PCR. Control reactions with actin
primers were used to control for minor differences in sam-
ple loading. Similar to the Northern analysis of total RNA,
the PCE-containing luc RNAs were less abundant in the
nucleoplasm and cytoplasm (Table 3, compare U3-Luc
with PCE-Luc, IE-U3-Luc with IE-PCE-Luc). Moreover, the
accumulation of PCE-Luc RNA in the cytoplasm was
lower by a factor of 3. Western analysis was used to verify
appropriate fractionation of the nucleoplasm and cyto-
plasm. Histone H1 was present exclusively in the nuclear
fractions and β-tubulin was present exclusively in the
cytoplasmic fractions (Figure 4). Immunoblotting with
actin verified equivalent sample loading among the sam-
ples. The results indicate that PCE-containing luc RNA
exhibits lower cytoplasmic accumulation and this differ-
PCE increases lentiviral vector transgene activity in trans-fected ellsFigure 2
PCE increases lentiviral vector transgene activity in
transfected cells. (A) Northern blot analysis of total RNA
that was isolated 48 hrs after transfection with the indicated
vector and hybridized with DNA probe complementary to
luc open reading frame or gapdh loading control. The gapdh
level was used to standardized minor differences in RNA
sample concentration. (B) Graphic representation of data
from (A) with white bars indicating the luc RNA levels stand-
ardized to gapdh loading control. Black bars indicate transla-
tional efficiency relative to the corresponding U3 vector.
Translational efficiency is defined as the ratio of Luc activity
in these samples to luc mRNA in (A).
4
3
2
1
0
l
u
c
R
N
A
(
x

1
0
5

p
h
o
s
p
h
o
r
i
m
a
g
e
r

u
n
i
t
s
)
L
u
c

p
r
o
t
e
i
n

r
e
l
a
t
i
v
e

t
o

l
u
c

R
N
A
Mock PCE U3 IE-PCE IE-U3
<MD
5
4
3
2
1
0
luc
gapdh
Mock PCE U3
Total RNA
A
B
IE-PCE IE-U3Page 4 of 10
(page number not for citation purposes)
cated that the combination of PCE and CMV-IE produced
a cumulative 4-fold increase in transgene protein produc-
ence is proportional to the reduction observed in steady
state luc mRNA.

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
To investigate the possibility that PCE reduced the stabil-
ity of the transgene mRNA, the transduced cells were
treated with actinomycin D for intervals between 0 and 18
hrs and total cellular RNA was subjected to the Northern
analysis. Similar to the Northern analysis shown in Figure
3, the PCE-containing luc RNAs exhibited lower steady
state levels compared to the PCE-lacking controls (com-
pare PCE and U3 in Figure 5A, compare IE-PCE and IE-U3
in Figure 5B). In contrast to the differences in luc tran-
script, the abundance of gapdh loading control was simi-
lar among the samples. As shown graphically in Figure 5C
and 5D, the decay kinetics of these PCE-containing luc
RNAs were no faster than the PCE-lacking control RNAs.
The results indicate that PCE did not reduce luc RNA sta-
bility. These results taken together with the RT-real time
PCR results indicate that the lower steady state level of
PCE-Luc RNA is not attributable to reduced RNA stability,
but to lower cytoplasmic accumulation. Comparison of
the level of Luc activity per molecule of luc RNA present
in the cytoplasm indicated that PCE increased Luc protein
yield 5 to 6-fold in transduced cells (Table 3). These
results indicate that despite the reduction of cytoplasmic
accumulation of PCE-luc RNA in transduced cells, PCE
translation enhancement activity was sustained. We con-
clude that the magnitude of translational enhancement is
similar in transfected and transduced cells.
Discussion
Work presented here shows that the PCE can stimulate an
increase in lentiviral vector transgene translation. This
activity of PCE functioned in synergy with a heterologous
transcriptional enhancer and produced a significant 11 to
17-fold increase in gene expression in transfected cells.
The presence of PCE is associated with a lower steady state
of the transgene mRNA in transduced 293 cells but is not
attributable to reduced RNA stability. It is generally
accepted that the abundance and localization of an mRNA
for this observation is that activity of an integrated pro-
moter in a transduced cell is modulated in relation to the
local chromatin structure. For example, Williams et al.
[23] found that binding of histone deacetylase enzyme
HDAC1 to the LTR of an HIV-1 provirus induced altera-
tions in the chromatin structure that disrupted binding of
RNA polymerase II and silenced transcription. Addition-
ally, Hofmann et al. showed that methylation of the pro-
moter of a lentiviral vector provirus led to transcriptional
inactivation [24]. A possible explanation for the lower
steady state level of PCE-Luc RNA in our transduced cells
is reduced transcription attributable to promoter methyl-
ation. A further consideration is that our Northern blot
and RT-real time PCR results indicate that the lower steady
state PCE-Luc RNA was attributable to post-transcrip-
tional modulation.
It is now clear that steps in transcriptional and post-tran-
scriptional control of gene expression are functionally and
physically linked [25]. For example, cotranscriptional
interaction with nuclear RNA processing factors is medi-
ated by the carboxy-terminal domain (CTD) of the largest
subunit of RNA polymerase II [26-29]. The CTD choreo-
graphs deposition of multiprotein complexes on nascent
pre-RNAs that implement efficient export from the
nucleus and translation in the cytoplasm [25,27,28]. The
multisubunit TREX complex, which is conserved from
yeast to man, links the apparently distinct processes of
transcription and mRNA export [30]. Biochemical analy-
sis of TREX has identified interaction with both intronless
and intron-containing genes and determined a relation-
ship between its cotranscriptional recruitment and pre-
mRNA retention [31]. Furthermore, the process of tran-
scription is linked with mRNA 3' end formation. RNA
polymerase II elongation complexes undergo multiple
transitions at the 3' end of genes [27,32,33]. An exchange
of elongation and polyadenylation/termination factors at
Table 2: The combination of PCE and CMV IE increased Luc activity in transduced cells
Luc activity (Relative Light Units)
a
Replicate Experiment
Vector HeLa CEM-A D17 293
U3-Luc 13,639 ± 2,150 (1)
b
17,100 ± 524 (1) 18,093 ± 349 (1) 15,099 ± 539 (1)
PCE-Luc 51,998 ± 3108 (3.8)* 52,372 ± 3,354 (3.0)* 50,412 ± 2,997 (2.7)* 27,294 ± 1,173 (1.8)*
IE-U3-Luc ND
c
ND ND 26,197 ± 95 (1.7)
IE-PCE-Luc ND ND ND 59,620 ± 286 (3.9)
a
Equivalent vector virus particles were measured by Gag p24 ELISA and 4 × 10
5
pg Gag was used to transduce the indicated target cell lines. Total
cellular protein was harvested 48 hrs post-transduction and equivalent protein was subject to Luciferase assay. Standard deviations were calculated
from results of two or more replicate experiments. ANOVA with repeated measures determined that increases in response to PCE were
significant, (p-value of 0.017).
b
(), Fold difference relative to U3-luc vector.Page 5 of 10
(page number not for citation purposes)
may be different when expressed from transfected DNA or
from an integrated vector in infected cells. An explanation
the 3' end of genes choreographs efficient transcription
termination and polyadenylation. Alteration in the tran-

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
scriptional activity of the promoter may invoke unex-
pected effects on 3' end formation and reduce the steady
state mRNA. Based on our results of Northern and RT-real
time PCR RNA analysis we speculate that incorporation of
the vector provirus into chromatin altered the cotranscrip-
tional deposition of nuclear factors on the nascent PCE-
Luc RNA that mediate efficient 3' end formation or
nuclear export. Our analysis also determined that the
stimulatory effect of PCE on translation activity was sus-
tained despite less efficient upstream steps in gene expres-
sion. This observation suggests that factors necessary for
PCE translation stimulation remained available despite
less cytoplasmic accumulation.
Our study using the luc transgene suggests that changes in
early steps in ribonucleoprotein particle formation pro-
foundly influenced the availability of mRNA available for
translation enhancement by PCE. We project that the
activity of PCE and CMV IE to co-ordinately stimulate pro-
tein output will be sustained in other transgenes. How-
ever, the unique features of any particular transgene s
vector integration is expected to induce epigenetic modu-
lation of gene transcription that may profoundly affect the
absolute level of RNA available for protein synthesis. The
results of our study are consistent with the recent realiza-
tion of tight linkage between the transcription and post-
transcriptional steps in gene expression and emphasize
the important role epigenetic modulation plays in vector
gene expression.
Conclusion
Coordinate enhancement of transgene transcriptional
and post-transcriptional expression represents a potent
approach to increase transgene protein production in a
broad array of gene expression systems, including lentivi-
ral vectors, other viral vectors and non-viral gene expres-
sion plasmids. Our results show that combined
introduction of the SNV PCE 5' terminal translational
enhancer and CMV IE transcriptional enhancer to HIV-1
based lentiviral vectors significantly improved protein
yield per molecule of intronless transgene RNA in trans-
fected cells and in four transduced cell lines. Increasing
the protein yield per RNA molecule is expected to be a use-
ful approach in a diversity of gene expression systems.
This approach could compensate for limited promoter
activity observed in some in vivo studies wherein strong
promoter activity was not achievable using a tissue-spe-
cific promoter [35].
Methods
Plasmid construction
The Luc vectors were derived from self inactivating
pHR'CMV-GFP [36]. First, we constructed HIVSIN-Luc by
insertion of a linker (5'TCGATGGATCCACTAGTC 3' and
5'TCGAGACTAGTGGATCCA 3') into the XhoI site in
pHR'CMV-GFP thereby introducing an SpeI site. GFP was
replaced with the luc open reading frame in the BamHI-
XbaI fragment from pCAM-Luc by ligation into BamHI
and SpeI. pCAM-Luc was constructed by insertion of a
PCR product containing luc from pGL3 (Promega, Madi-
son, WI) with BamHI and XbaI termini into pPCR-Script
CAM SK+ (Stratagene, La Jolla, CA). PCE-Luc was derived
from HIVSIN-SNVLTR-GFP and U3-Luc was derived from
HIVSIN-SNVU3-GFP. To construct HIVSIN-SNVLTR-GFP
and HIVSIN-SNVU3-GFP, the NdeI-BamHI fragment
from pHR'CMV-GFP was replaced with the NdeI-BamHI
fragment that contains the SNV U3RU5 or U3 sequences
of pYW100 and pYW205 [15], respectively. In order to
replace the GFP fragment in HIVSIN-SNVLTR-GFP and
HIVSIN-SNVU3-GFP with the luc open reading frame,
DNA oligonucleotides (KB973–KB974) were annealed
and ligated a the XhoI site in each vector and then NdeI-
SpeI fragment that contains SNV U3RU5-Luc and SNV
RU5-Luc from pSNVRU5luc and pSNVluc [17] was
PCE increases translational efficiency of lentiviral vector transgen RNA in tr nsduced ellsFigur 3
PCE increases translational efficiency of lentiviral
vector transgene RNA in transduced cells. (A) North-
ern blot analysis of total cellular RNA that was isolated 48
hours post-transduction and hybridized with a probe com-
plementary to luc or gapdh loading control (B) Graphic rep-
resentation of the luc RNA in transduced cells. Black bars
indicate translational efficiency relative to the corresponding
U3 vector. Translational efficiency is defined as the ratio of
Luc activity to luc mRNA.
luc
Mock PCE U3 IE-PCE IE-U3
A
B
gapdh
l
u
c
R
N
A
(
x

1
0
5

p
h
o
s
p
h
o
r
i
m
a
g
e
r

u
n
i
t
s
)
L
u
c

p
r
o
t
e
i
n

r
e
l
a
t
i
v
e

t
o

l
u
c

R
N
A
4
3
2
1
70
60
40
20
1
Mock PCE U3 IE-PCE IE-U3
<MDPage 6 of 10
(page number not for citation purposes)
likely to influence the efficiency of 3' end formation or
other post-transcriptional process [33,34]. Furthermore,
inserted to create PCE-Luc and U3-Luc, respectively. The
CMV-IE enhancer region from pRL-CMV (Promega, Mad-

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
ison, WI) was amplified by PCR with primers
(5'TTTTTATCGATAAGCTCAATATTGGCCATATTATTCAT
TGG3' and
5'TTTTCATATGCAGTTGTTACGACATTTTGGAAAG3')
and ligated with NdeI-ClaI-digested PCE-Luc and U3-Luc
in order to create IE-PCE-Luc and IE-U3-Luc, respectively.
Transient transfection and Luciferase assay
Transient transfections were performed on 2 × 10
5
293
cells in duplicate 60 mm plates. Five µg vector DNA and
0.5 µg pRL-CMV (Promega, Madison, WI) were cotrans-
fected by CaPO
4
method [15]. The cells were harvested in
PBS at 48 h post transfection, centrifuged at 1500 × g for
3 min and resuspended in 150 µl of ice-cold NP-40 lysis
buffer (20 mM TrisHCl [pH 7.4], 150 mM NaCl, 2 mM
EDTA, and 1% NP-40). Dual-Luciferase reporter assay
(Promega, Madison, WI) was performed with 10 µl lysate,
100 µl Luciferase assay reagent II (Promega, Madison, WI)
and 100 µl Stop&Glow™ (Promega, Madison, WI) accord-
ing to manufacturer's protocol and quantified in a Lumi-
count luminometer (Packard Instrument Company Inc.,
Downers Grove, IL). The level of Ren activity was used to
standardize transfection efficiency. Luc activity is pre-
sented relative to Ren activity. Luc activity in transduced
cells was also determined 48 hours after transduction by
the same procedure. Cells were lysed in 100 µl ice-cold
NP-40 lysis buffer and Luc assay was performed with 10 µl
lysate and 100 µl Luciferase assay reagent (Promega, Mad-
ison, WI).
Preparation of vector virus stocks and transduction
Lentivirus vector stocks were produced by transient triple
transfection of 293T cells with 10 µg of HIV-1 gag-pol
packaging plasmid pCMV∆R8.2 [37], 2 µg of the pMD.G
VSV glycoprotein expression plasmid [37], and 10 µg of
the cells were cultured in fresh DMEM (Invitrogen, CA),
10% FBS and 10 mM sodium butyrate for 8 hours. The
supernatants were collected at 12 hour intervals over a 60
hour time period and passed through a 0.2-µm filter
(Corning, NY) and concentrated by ultracentrifugation at
80,000 × g at 24°C for 2.5 h in a Beckman SW28 rotor.
HIV-1 Gag concentration was determined by Gag p24
ELISA (Coulter, Hialeah, FL). 293, HeLa, CEMx174 and
D17 cells were transduced with 4 × 10
5
pg Gag in 6-well
plates by spinoculation at 1500 × g for one hour at 32°C
[38]. Spinoculation of 293 cells was performed in the
presence of 8 ug/ul polybrene.
RNA preparation
Total RNA was isolated from approximately 5 × 10
5
cells
in 0.5 ml Trizol reagent (Invitrogen, CA) according to
manufacturer's protocol. Cells were treated with 5 ug/ml
Western blots demonstrate appropriate subcellular fraction-ation of transduced cellsFigure 4
Western blots demonstrate appropriate subcellular
fractionation of transduced cells. Equivalent amounts of
each nuclear or cytoplasmic fraction were subjected to
immunoblot with antiserum against the nuclear protein his-
tone H1; the cytoplasmic protein β-tubulin; and loading con-
trol β-actin, which is distributed in the nucleus and
cytoplasm. The results determined that similar levels of pro-
tein were loaded and verified effective subcellular fractiona-
B-tubulin
B-actin
Histone H1
U3
Nucleoplasm Cytoplasm
PCE
IE-
U3
IE-
PCE U3PCE
IE-
U3
IE-
PCE
Table 3: PCE correlates with reduced cytoplasmic accumulation of luc RNA in transduced cells.
RNA copy number (× 10
3
)
a
Nucleoplasm Cytoplasm Cytoplasmic
accumulation
b
Translational
efficiency
c
Vector luc actin luc actin
U3-Luc 56.7 ± 5.4 (1) 59.3 25.3 ± 2.8 (1) 375 0.45 1
PCE-Luc 34.9 ± 3.8 (0.47) 76.6 5.4 ± 0.2 (0.2) 372 0.16 5
IE-U3-Luc 91.3 ± 1.5 (1) 69.1 46.8 ± 12.9 (1) 417 0.51 0.5
IE-PCE-Luc 96.8 ± 2.7 (0.88) 82.5 18.3 ± 2.7 (0.4) 384 0.19 3
a
Equivalent amounts (100 ng) of RNA from either the nucleoplasm or cytoplasm were reverse transcribed to generate cDNA and one-tenth of
each reaction was quantified by real-time PCR with primers specific to luc or actin. Copy numbers were derived from standard curves generated
with pGL3 luciferase plasmid in the range of 10
1
to 10
9
copies. Reactions were performed in duplicate and the mean and standard deviation are
indicated. (), levels of luc normalized to actin relative to PCE-lacking controls, U3-Luc or IE-U3-Luc, respectively.
b
Ratio of the copy number of luc RNA in the cytoplasm and nucleoplasm.
c
Ratio of Luc activity to copy number of cytoplasmic luc RNAPage 7 of 10
(page number not for citation purposes)
the vector plasmid by the CaPO
4
method [15]. After over-
night transfection of 5 × 10
6
293T cells in a 10-cm plate,
tion.

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
Page 8 of 10
(page number not for citation purposes)
The half-life of luc RNA is not decreased by PCEFigure 5
The half-life of luc RNA is not decreased by PCE. 293 cells transduced with PCE-Luc, U3-Luc, IE-PCE-Luc or IE-U3-Luc
were treated with actinomycin D (ActD) for indicated time intervals and total RNA was isolated and subjected to Northern
blot analysis with luc or gapdh complementary DNA probes. (A,B) Northern blot results from a representative of two repli-
cate experiments. The abundance of PCE-containing RNAs is lower than PCE-lacking RNAs, while the abundance of gapdh
loading control was similar. (C,D) Decay curves were generated with luc RNA signal standardized to gapdh. The presence of
PCE did not reduce the stability of luc RNA.
PCE U3
0 2 4 6 12 18 0 2 4 6 12 18hrs ActD
luc
gapdh
IE-PCE IE-U3
02461218 02461218hrs ActD
luc
gapdh
PCE
0
5
10
15
20
25
30
ActD hours
P
I

u
n
i
t
s

x
1
0
3
P
I

u
n
i
t
s

x
1
0
3
U3
20
40
60
80
100
120
140
160
180
ActD hours
0246 18120246 1812
IE-PCE
0
50
100
150
200
250
0 2 4 6 12 18
ActD hours
P
I

u
n
i
t
s

x
1
0
3
P
I

u
n
i
t
s

x
1
0
3
IE-U3
0
50
100
150
200
250
300
350
0246 1812
ActD hours
A
C
B
D

Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
actinomycin D for 2, 4, 6, 12 and 18 hours. To harvest
nuclear and cytoplasmic RNA, a subconfluent 100 mm
plate of cells was incubated with hypotonic lysis buffer
(10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl,
0.5%NP40, and 0.5 mM dithiothreitol) for 10 min on ice
andsubjected to two rounds of centrifugation at 3000 × g
for 2 mins at 4°C. One-tenth of the nuclear pellet and
cytoplasmic supernatant were reserved for Western blot-
ting. The pellet was treated with Trizol (Invitrogen, CA)
and the supernatant was treated with Trizol-LS (Molecular
Research Center, Cincinnati, OH) and RNA was extracted
by the manufacturer's protocol.
RNA analysis
For Northern blot analysis, 5 µg total RNA was separated
on 1.2% agarose gels containing 5% formaldehyde, trans-
ferred to Duralon-UV membrane (Stratagene, La Jolla,
CA), and incubated with either luc or gapdh DNA probes.
The probes were prepared by a random-primer DNA-labe-
ling system (Invitrogen, CA) with gel purified luc or
gapdh restriction products and [α-
32
P]dCTP. The hybridi-
zation products were scanned with PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) and quantified by
ImageQuant software (Molecular Dynamics, Sunnyvale,
CA). Sucrose gradients were prepared from 1 × 10
7
293
cells in a T150 flask 48 hours post-transfection as
described previously [17] and isolated RNA was subjected
to Northern blot.
For reverse transcription, random hexamer and Sensis-
cript reverse transcriptase (Qiagen, Germany) were used
to generate cDNA from 100 ng of cytoplasmic or nuclear
RNA. Ten percent of the cDNA preparation was used for
real-time PCR with primers complementary to luciferase
or actin and Quantitect SYBR Green PCR (Qiagen, Ger-
many) in a Lightcycler (Roche, Germany). Copy numbers
were derived from standard curves generated with pGL3
luciferase plasmid in the range of 10
1
to 10
9
copies. Reac-
tions were performed in duplicate and the mean and
standard deviation are presented.
Western blotting
Bradford assay was used to measure 50 µg of protein from
nuclear and cytoplasmic fractions. Proteins were sepa-
rated by SDS-PAGE and transferred to nitrocellulose
membrane. Immunoblotting was performed with mouse
monoclonal antibodies against histone H1, β-tubulin and
β-actin (Abcam, Cambridge, MA). Visualization was per-
formed with Luminol reagent (Santa Cruz Biotechnology,
Santa Cruz, CA).
Competing interests
The author(s) declare that they have no competing inter-
Authors' contributions
AY conceived of the study, carried out the vector construc-
tion, experimental evaluation, and participated in the
data analysis and preparation of the manuscript. SF partic-
ipated in the design of the study and carried out the statis-
tical analysis. MDL participated in the preparation of the
manuscript. KBL coordinated the design and implementa-
tion of the study, the data analysis, and the preparation of
the manuscript. All authors read and approved the final
manuscript.
Acknowledgements
This work was supported by National Institutes of Health National Cancer
Institute Comprehensive Cancer Center grant P30 CA16058 and Program
Project grant P01 CA100730. We thank Shuiming Qian for assistance with
real time PCR and Tiffiney Roberts Hartman, Kate Hayes, Cheryl Bolinger,
Nicole Placek and Shuiming Qian for critical comments on the manuscript.
We are grateful to Tim Vojt for illustration and formatting.
References
1. Ferrari G, Salvatori G, Rossi C, Cossu G, Mavilio F: A retroviral
vector containing a muscle-specific enhancer drives gene
expression only in differentiated muscle fibers. Hum Gene Ther
1995, 6:733-742.
2. Vile RG, Diaz RM, Miller N, Mitchell S, Tuszyanski A, Russell SJ: Tis-
sue-specific gene expression from Mo-MLV retroviral vec-
tors with hybrid LTRs containing the murine tyrosinase
enhancer/promoter. Virology 1995, 214:307-313.
3. Grande A, Piovani B, Aiuti A, Ottolenghi S, Mavilio F, Ferrari G: Tran-
scriptional targeting of retroviral vectors to the erythroblas-
tic progeny of transduced hematopoietic stem cells. Blood
1999, 93:3276-3285.
4. Zhao-Emonet JC, Marodon G, Pioche-Durieu C, Cosset FL, Klatz-
mann D: T cell-specific expression from Mo-MLV retroviral
vectors containing a CD4 mini-promoter/enhancer. J Gene
Med 2000, 2:416-425.
5. Gelinas C, Temin HM: Nondefective spleen necrosis virus-
derived vectors define the upper size limit for packaging
reticuloendotheliosis viruses. Proc Natl Acad Sci U S A 1986,
83:9211-9215.
6. Verma IM, Somia N: Gene therapy -- promises, problems and
prospects. Nature 1997, 389:239-242.
7. Braddock M, Muckenthaler M, White MR, Thorburn AM, Sommerville
J, Kingsman AJ, Kingsman SM: Intron-less RNA injected into the
nucleus of Xenopus oocytes accesses a regulated translation
control pathway. Nucleic Acids Res 1994, 22:5255-5264.
8. Matsumoto K, Wassarman KM, Wolffe AP: Nuclear history of a
pre-mRNA determines the translational activity of cytoplas-
mic mRNA. EMBO J 1998, 17:2107-2121.
9. Choi T, Huang M, Gorman C, Jaenisch R: A generic intron
increases gene expression in transgenic mice. Mol Cell Biol
1991, 11:3070-3074.
10. Gudikote JP, Imam JS, Garcia RF, Wilkinson MF: RNA splicing pro-
motes translation and RNA surveillance. Nat Struct Mol Biol
2005, 12:801-809.
11. Wiegand HL, Lu S, Cullen BR: Exon junction complexes mediate
the enhancing effect of splicing on mRNA expression. Proc
Natl Acad Sci U S A 2003, 100:11327-11332.
12. Nott A, Le Hir H, Moore MJ: Splicing enhances translation in
mammalian cells: an additional function of the exon junction
complex. Genes Dev 2004, 18:210-222.
13. Nott A, Meislin SH, Moore MJ: A quantitative analysis of intron
effects on mammalian gene expression. RNA 2003, 9:607-617.
14. Lu S, Cullen BR: Analysis of the stimulatory effect of splicing on
mRNA production and utilization in mammalian cells. RNA
2003, 9:618-630.
15. Butsch M, Hull S, Wang Y, Roberts TM, Boris-Lawrie K: The 5' RNAPage 9 of 10
(page number not for citation purposes)
ests.
terminus of spleen necrosis virus contains a novel posttran-
scriptional control element that facilitates human immuno-

Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
Retrovirology 2006, 3:13 http://www.retrovirology.com/content/3/1/13
deficiency virus Rev/RRE-independent Gag production. J Virol
1999, 73:4847-4855.
16. Hull S, Boris-Lawrie K: 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 2002, 76:10211-10218.
17. Roberts TM, Boris-Lawrie K: The 5' RNA terminus of spleen
necrosis virus stimulates translation of nonviral mRNA. J
Virol 2000, 74:8111-8118.
18. Roberts TM, Boris-Lawrie K: Primary sequence and secondary
structure motifs in spleen necrosis virus RU5 confer transla-
tional utilization of unspliced human immunodeficiency
virus type 1 reporter RNA. J Virol 2003, 77:11973-11984.
19. Dangel AW, Hull S, Roberts TM, Boris-Lawrie K: Nuclear interac-
tions are necessary for translational enhancement by spleen
necrosis virus RU5. J Virol 2002, 76:3292-3300.
20. Hirano A, Wong T: Functional interaction between transcrip-
tional elements in the long terminal repeat of reticuloen-
dotheliosis virus: cooperative DNA binding of promoter- and
enhancer-specific factors. Mol Cell Biol 1988, 8:5232-5244.
21. Ridgway AA, Kung HJ, Fujita DJ: Transient expression analysis of
the reticuloendotheliosis virus long terminal repeat ele-
ment. Nucleic Acids Res 1989, 17:3199-3215.
22. Sheay W, Nelson S, Martinez I, Chu TH, Bhatia S, Dornburg R:
Downstream insertion of the adenovirus tripartite leader
sequence enhances expression in universal eukaryotic vec-
tors. Biotechniques 1993, 15:856-862.
23. Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene
WC: NF-kappaB p50 promotes HIV latency through HDAC
recruitment and repression of transcriptional initiation.
EMBO J 2006, 25:139-149.
24. Hofmann A, Kessler B, Ewerling S, Kabermann A, Brem G, Wolf E,
Pfeifer A: Epigenetic regulation of lentiviral transgene vectors
in a large animal model. Mol Ther 2006, 13:59-66.
25. Maniatis T, Reed R: An extensive network of coupling among
gene expression machines. Nature 2002, 416:499-506.
26. Neugebauer KM: On the importance of being co-transcrip-
tional. J Cell Sci 2002, 115:3865-3871.
27. Proudfoot NJ, Furger A, Dye MJ: Integrating mRNA processing
with transcription. Cell 2002, 108:501-512.
28. Reed R, Hurt E: A conserved mRNA export machinery cou-
pled to pre-mRNA splicing. Cell 2002, 108:523-531.
29. Jensen TH, Dower K, Libri D, Rosbash M: Early formation of
mRNP: license for export or quality control? Mol Cell 2003,
11:1129-1138.
30. Strasser K, Masuda S, Mason P, Pfannstiel J, Oppizzi M, Rodriguez-
Navarro S, Rondon AG, Aguilera A, Struhl K, Reed R, Hurt E: TREX
is a conserved complex coupling transcription with messen-
ger RNA export. Nature 2002, 417:304-308.
31. Abruzzi KC, Lacadie S, Rosbash M: Biochemical analysis of TREX
complex recruitment to intronless and intron-containing
yeast genes. EMBO J 2004, 23:2620-2631.
32. Ahn SH, Kim M, Buratowski S: Phosphorylation of serine 2 within
the RNA polymerase II C-terminal domain couples tran-
scription and 3' end processing. Mol Cell 2004, 13:67-76.
33. Kim M, Ahn SH, Krogan NJ, Greenblatt JF, Buratowski S: Transitions
in RNA polymerase II elongation complexes at the 3' ends of
genes. EMBO J 2004, 23:354-364.
34. Huertas P, Aguilera A: Cotranscriptionally formed DNA:RNA
hybrids mediate transcription elongation impairment and
transcription-associated recombination. Mol Cell 2003,
12:711-721.
35. Gerdes CA, Castro MG, Lowenstein PR: Strong promoters are
the key to highly efficient, noninflammatory and noncyto-
toxic adenoviral-mediated transgene delivery into the brain
in vivo. Mol Ther 2000, 2:330-338.
36. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM: Develop-
ment of a self-inactivating lentivirus vector. J Virol 1998,
72:8150-8157.
37. Naldini L, Blomer U, Gage FH, Trono D, Verma IM: Efficient trans-
fer, integration, and sustained long-term expression of the
transgene in adult rat brains injected with a lentiviral vector.
Proc Natl Acad Sci U S A 1996, 93:11382-11388.
38. Bahnson AB, Dunigan JT, Baysal BE, Mohney T, Atchison RW, Nim-
retroviral mediated gene transfer. J Virol Methods 1995,
54:131-143.yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Page 10 of 10
(page number not for citation purposes)
gaonkar MT, Ball ED, Barranger JA: Centrifugal enhancement of