High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site
N.
Ramesh
,
Seung-Taik
Kim
+
,
Ming Q.
Wei
,
Mehraneh
Khalighi
and
William R. A.
Osborne*
Department of Pediatrics, University of Washington School of Medicine,
Seattle
, WA 98195,
USA
Received April 2, 1996;
Revised and Accepted June 3, 1996
ABSTRACT
Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES)
followed by the coding region of
[beta]-galactosidase (
[beta]-gal) or therapeutic genes, with the selectable neomycin phosphotransferase
gene under the control of the viral long terminal repeat (LTR) promoter. LNFX,
a vector with a multiple cloning site 3'
to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat
erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and
[beta]-gal. In transduced primary rat vascular smooth muscle cells the cytokines
were expressed at high levels, similar to those obtained from vectors employing
the viral LTR promoter. LNFZ, a vector encoding
[beta]-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector
containing the poliovirus (Po) internal ribosome entry site. Primary canine
vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar
activities of [beta]
-gal and neomycin phosphotransferase (NPT). Overall, these vectors had
titers between 106
and 2*107
c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.
INTRODUCTION
Eukaryotic protein synthesis begins with the binding of initiation factors and
the 40S ribosomal subunit to the 5'-capped structure at the 5' terminus of the mRNA, followed by the scanning of this
complex and translation initiation at the most proximal AUG codon in a
favorable context (
1
). In contrast, picornavirus RNAs are not capped and the translation initiation
depends on direct binding of initiation complexes to the internal ribosomal
entry site (IRES) within the 5' non-translated region (
2
-
8
). Based on their IRES sequence the picornaviruses can be divided into three
groups: entero- and rhinoviruses, cardio- and aphthoviruses, and hepatitis A virus (
9
,
10
). Translation studies in a cell-free system showed IRES from the three groups responded differently,
suggesting that the functional requirements for efficient IRES activity vary
widely (
11
).
Retroviral vectors may be designed using these IRES to express multiple proteins
from a single mRNA (
12
-
14
). We have shown the stable expression of both genes in bicistronic mRNAs transcribed from retroviral vectors in which encephalomyocarditis virus (EMCV) or poliovirus (Po) IRES were inserted between two cDNA coding
regions (
12
). Foot-and-mouth disease virus (FMDV) is an aphthovirus of the picornavirus
family and contains an IRES of ~480 bp (
15
,
16
), smaller than the 730 bp of the IRES of EMCV or Po (
4
,
6
,
12
). In this study we tested the ability of retroviral vectors containing FMDV
IRES to provide high-titer and high-level bicistronic gene expression in transduced primary cells.
MATERIALS AND METHODS
Cell culture
PE 501 ecotropic and PA317 amphotropic packaging cells (
17
) and rat and dog vascular smooth muscle cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Canine vascular
smooth muscle cells were obtained from peripheral veins by enzymatic digestion
as described for rat vascular tissue (
18
).
Retroviral vector construction
Scale drawings of the vectors with their best titers are shown in Figure
1
. Vector names are based on the order of genetic elements within the construct.
Abbreviations and symbols are: LTR, retroviral long terminal repeat; S, simian
virus 40 early promoter and enhancer; Neo, bacterial neomycin
phosphotransferase cDNA; F, FMDV 5' non-translated IRES; Po, poliovirus IRES; E, EMCV IRES; [beta]-gal, [beta]-galactosidase cDNA; A, human adenosine
deaminase cDNA; rEP, rat erythropoietin cDNA; rG, rat granulocyte colony
stimulating factor cDNA. LNFZ vector construction:
Sac
I fragment of FMDV IRES (
16
) from pBIR (plasmid kindly provided by Dr E. Martinez-Salas, Madrid) was inserted into the
Sac
I site of pGEM-1. Po IRES from LNPoZ (
12
) was removed by
Bst
BI and
Avr
II digestion and was replaced by
Acc
I and
Eco
RI fragment of FMDV IRES after gap filling. LNFX was made by inserting the FMDV
fragment from LNFZ in place of the SV40 promoter in the vector LNSX (
17
). The
Sac
I fragment from LNFX was cloned into pUC19
Sac
I site, cut out with
Sma
I and
Eco
RI and cloned into pBSK in the same sites to make pBSK-F, which was then cut with
Bam
HI and
Cla
I and used to replace SV40 in LNSX. LNFrEP was obtained by inserting rat Epo
cDNA [kindly provided by Drs Boissel and Bunn (
19
)] into pBSK-F and inserting the FrEP fragment in place of SV40 in LNSX. LNFA was
constructed by cloning the
Cla
I fragment of LNCA (
20
) encoding ADA cDNA into the
Cla
I site of LNFX. Amphotropic retroviral vectors were produced by shuttle packaging using PE501 and PA317 packaging cells (
17
). Briefly, plasmids were introduced into PE501 cells by calcium phosphate-mediated transfection, virus harvested 2 days later and used to infect
PA317 cells. Clonal cell lines were isolated following selection in 1 mg/ml
G418 and ~10 infected clones for each vector were screened for high-titer virus production by assay of secreted virus on NIH 3T3 TK
-
cells. Target cells were exposed to virus overnight, split 1 to 10 and drug
resistant colonies counted after 5 days culture. Producer clones that contained
single unrearranged proviruses by Southern analysis (
21
) were chosen for study.
Enzyme activities
Activities of [beta]-gal in cell extracts were determined by spectrophotometric assay
using 14 mM o-nitrophenyl [beta]-o-galactopyranoside as a substrate in 0.3 M sodium
phosphate pH 7.0 (
22
). Neomycin phosphotransferase activities were measured by [[gamma]-
32
P]ATP phosphate transfer to neomycin (
23
).
Cytokine assays
Rat erythropoietin was determined by measuring proliferation of HCD 57, a murine
cell line responsive to this cytokine (
18
,
24
) and rat G-CSF was assayed by proliferation of NSF-60, a murine cell line responsive to G-CSF (
25
). Recombinant human cytokines, kindly supplied by Amgen, were used to construct
standard curves.
RESULTS
Virus production
Amphotropic helper-free virus was made from PA317 packaging cells, and G418 resistant clones
that contained unrearranged proviruses were chosen for further study (data not
shown). The five highest titers obtained from each new construct showed a
reasonably tight range, and for all vectors the best two or three titers were
comparable (Table
1
). We compared LNFZ with LNPoZ, a similar vector that contains an IRES from
poliovirus (
12
). For LNFZ-PA317 packaging cells the best viral titer was 10
6
c.f.u./ml whereas for LNPoZ-PA317 packaging cells a viral titer of 10
5
c.f.u./ml was achieved (Fig.
1
). The titer of LNFA was ~3-fold greater than LAEN (
12
), a similar vector containing EMCV IRES (Fig.
1
). Overall, the highest virus titers of the FMDV-constructs encoding therapeutic genes were in the range of 9 * 10
6
to 2 * 10
7
c.f.u./ml (Table
1
), which are equivalent to or higher than the highset titers from LrGSN and
LrEPSN, similar two gene vectors employing internal promoters (Fig.
1
).
Analysis of enzyme activities in transduced primary cells
Quantification of vector encoded protein expression obtained from extracts of
packaging cells does not provide an accurate measure of transduced primary cell
expression. Therefore, primary canine smooth muscle cells were transduced,
selected in G418 and assayed for [beta]-gal (
22
) and neo activity (
23
). Extracts from LNFZ and LNPoZ transduced vascular smooth muscle cells showed
similar [beta]-gal and neo activities (Table
2
). X-gal staining for [beta]-gal activity showed uniform staining with both the vectors
(data not shown).
Enzyme activities in transduced canine vascular smooth muscle cells
Virus
Level of enzyme
a
[beta]-gal
NPT
None
<0.1
<0.1
LNFZ
2.4
25.9
LNPoZ
2.6
31.9
a
[beta]-galactosidase units are expressed as [mu]mol/min/mg protein. NPT units are c.p.m. * 10
-3
/h/[mu]g protein.
Erythropoietin and granulocyte colony-stimulating factor expression
Primary rat vascular smooth muscle cells were infected with vectors encoding
either rat Epo cDNA or rat G-CSF cDNA, selected in G418, and assayed for the relevant cytokines. We
compared the cytokine expression of bicistronic two gene vectors using foot-and-mouth disease virus IRES (LNFrG and LNFrEP) with conventional two
gene vectors employing an internal SV40 viral promoter (LrGSN and LrEPSN)
(Table
3
). The vectors encoding erythropoietin produced equivalent activities in smooth
muscle cells from both the LTR promoter in LrEPSN, and the bicistronic IRES
construct in LNFrEP. However, neomycin phosphotransferase synthesis was not the
same from these constructs, with the retroviral LTR promoter in LNFrEP producing greater enzyme expression than the SV40 early promoter in LrEPSN (Table
3
), in agreement with previous studies (
12
). Secretion of G-CSF was greater from the LTR promoter in LrGSN than that observed from the
bicistronic LNFrG construct, unlike the equivalent levels of erythropoietin
obtained from LrEPSN and LNFrEP.
.
Cytokine expression in transduced rat vascular smooth muscle cells
Gene expression
a
Virus
Cytokine
NPT
LNFrEP
6.8
45.6
LrEPSN
6.7
8.7
LNFrG
0.1
ND
LrGSN
0.4
ND
a
NPT units same as in Table 2. Epo measured as mU/24 h/10
5
cells; G-CSF units are ng/24 h/10
6
cells. ND, not done.
DISCUSSION
These studies have shown that the IRES from foot-and-mouth disease virus allows the production of two-gene vectors that provide both high titer and high level gene
expression. The FMDV-containing vectors, encoding erythropoietin or G-CSF, both showed higher titers than the two-gene vectors employing conventional internal promoters that
were used as controls. However, the two constructs we tested for erythropoietin
synthesis exhibited similar activities in primary smooth muscle cells, whereas
the comparable vectors we tested for G-CSF secretion gave higher levels from the LTR promoter in comparison with the bicistronic construct. The unpredictability of vector
performance has been recognized (
26
), and suggests that vectors need to be constructed and tested in desired cell
types to determine their optimal activity.
The vector LNFZ encoding [beta]-gal and containing FMDV IRES produced a 10-fold increase in viral titer over LNPoZ, the virus containing
Po IRES. This increase in titer may indicate a higher efficiency of packaging
in viruses containing FMDV IRES. The relatively lower titer of vectors encoding
bacterial [beta]-gal (10
5
-10
6
c.f.u./ml) probably indicates RNA instability or a secondary structure not
optimal for efficient virus packaging. However, in a study of retroviral
vectors expressing human purine nucleoside phosphorylase (PN) we observed that
the presence or absence of an EMCV IRES did not produce a major change in viral
titer (
12
). In this study three viruses expressing human purine nucleoside phosphorylase,
LPNSN-2, LNEPN and LNPN had titers ranging from 3 * 10
6
to 5 * 10
6
c.f.u./ml. LPNSN-2 contains SV40 internal promoter, LNEPN contains EMCV IRES, and LNPN was
a control virus lacking both an internal promoter and an IRES.
Our data are in agreement with the recent studies of Borman
et al
. (
11
) that showed not only a wide range in the efficiency of internal initiation,
but also a higher efficiency from cardio- and aphthovirus IRES over poliovirus IRES. On the basis of IRES-initiated translation
in vitro
, the picornaviruses can be classified in a similar manner to their sequence
homologies, and that while all entry sites can direct internal ribosome entry,
the functional requirements for efficient IRES activity vary quite widely.
Our results with cDNA inserts of ~1 kb (rat granulocyte-colony stimulating factor, rat erythropoietin and human adenosine
deaminase) showed vector titers of ~10
7
c.f.u./ml. The five highest titers obtained for each constuct showed a
relatively close range, and at least two equivalently high titers were observed
for each, indicating that FMDV IRES viruses provide routinely higher titers
than vectors encoding Po or EMCV IRES (
12
). Increases in viral titer in constructs containing FMDV IRES was observed even
when using the 3 kb [beta]-gal cDNA insert, suggesting that this IRES may have a general
application in the construction of high titer retroviruses. As [beta]-gal expressing vectors are widely used, not only in gene therapy
applications but also in developmental studies, LNFZ offers a distinct
improvement over current viruses. Two gene retroviral vectors containing
picornavirus internal ribosome entry sites possess the distinct advantage that
selection for expression of one of the genes ensures expression of the other,
which is not the situation when other strategies are used (
8
,
12
,
13
). Finally, the low sequence homology between the FMDV IRES and the EMCV or Po
IRES together with its smaller size should make this IRES segment an ideal
candidate in the construction of useful tricistronic retroviral vectors.
ACKNOWLEDGEMENTS
We thank Dr E. Martinez-Salas for the plasmid pBIR encoding foot-and-mouth disease virus IRES. We thank Stella Lau for expert
technical assistance. This work was supported by grants DK 43727 and DK 38531
from the NIH.
