24
144 Chapter 5: Construction of subtype C reporter vectors: Potential use in screening of HIV-1 transcription modulators
144
Chapter 5: Construction of subtype C
reporter vectors: Potential use in screening of
HIV-1 transcription modulators
145
5.1 Introduction Human Immunodeficiency Virus type 1 (HIV-1) long terminal repeat (LTR)
promoter regulates viral gene expression by binding to viral and cellular factors
(Pereira et al, 2000). HIV-1 LTR, around six hundred fifty bases in length is
divided in to three regions: U3 (position - 454 to – 1 relative to the transcription
start site), R (position + 1 to + 60) and U5 (position + 60 to + 181). The analysis
of reporter gene expression placed downstream of the viral LTR promoter
sequences in a mammalian expression vector is widely used for understanding
HIV-1 LTR driven gene expression. When viral Tat gene is expressed along with
LTR-reporter gene construct, it substantially increases reporter gene activity.
Upon transcription, the ‘R’ region transcript folds into TAR hairpin structure that
interacts with viral Tat protein and leads to many fold increase in HIV-1
transcription (Brigati et al, 2003). Thus it provides a model system to study Tat
induced LTR mediated transcription and also to screen compounds, which
modulate viral transcription (Daelemans et al, 2001). Many viral and cellular
factors that regulate LTR promoter has been identified using such reporter gene
constructs in transient transfection assays (Ramirez et al, 2005). They have also
been used in studies related to basal activity of LTR promoter (Schwartz et al,
1990), HIV-1 inhibition (Yamamoto et al, 2002) and latency (Tobiume et al,
2002). Co-transfection of HIV-1 molecular clone along with LTR-reporter gene
construct provides a sensitive and quantitative measure of virus infection and
replication. Furthermore integrated copies of LTR-reporter gene construct(s) in
cell lines are useful and cost effective method for visualization and quantitation of
progression of HIV-1 infection and screening of anti-HIV-1 compounds (Gervaix
et al, 1997; Miyake et al, 2003; Princen et al, 2004).
5.1.1 Reporter genes and HIV-1 reporter cell lines
The Firefly luciferase (Luc) is most widely used quantitative reporter gene due to
its high sensitivity and relatively simple, well standardized assay procedure
(Gould et al, 1988). The green fluorescent protein (GFP) of the bioluminescent
146
jellyfish Aequorea victoria is also a commonly used reporter gene because of its
unique feature of green light emission after exposure to ultraviolet light without
extrinsic labeling or fixation (Chalfie et al, 1994; Yu et al, 2003). In addition,
GFP requires no cofactor and substrates. The fluorescence intensity of GFP is a
direct measurement of promoter activity, which can be easily observed by
fluorescence microscopy. Enhanced-GFP (EGFP) is a highly fluorescent and
stable mutant (S65T) of GFP. Chloremphenicol Acetyl Transferase (CAT), β-
galactosidase (β-gal), Secretory Alkaline Phosphatase (SEAP) are some other
reporter genes currently in use (Alam et al, 1990).
After identification of HIV-1 virus more than twenty years back, most of the
research has been performed with subtype B virus, which was the most prevalent
subtype till recent times. Many HIV-1 subtype B LTR-reporter gene construct
containing cell lines with reporter gene CAT (Felber et al,1988), GFP (Gervaix et
al,1997), Luciferase (Koseki et al,1998), β-galactosidase (Kimpton et al,1992),
Secretory Alkaline Phosphatase (SEAP) (Miyake et al, 2003) have been reported
and extensively used (Table-5.1). Subtype C has currently become the most
prevalent isolate worldwide. In recent studies related to comparison of LTR of
different HIV-1 subtypes, subtype C LTR-Luc constructs have been used
(Naghavi et al, 1999; Jeeninga et al, 2000). However, to the best of our
knowledge no subtype C LTR regulated GFP-Luciferase dual reporter vector has
been reported till date.
In the present work, we have used 5' LTR from pIndie-C1, a subtype C Indian
isolate to construct a dual reporter vector pLTRC-Luc-EGFP expressing both
Luciferase and EGFP under the control of LTR promoter in addition to two other
single reporter vectors, pLTRC-EGFP expressing EGFP and pLTRC-Luc
expressing Luciferase under the regulation of subtype C LTR. The plasmid maps
and construction scheme of single and dual reporter vectors are presented in
Figure-5.1. These subtype C reporter vectors are functionally active and are
useful in subtype C HIV-1 LTR promoter studies. These vectors could also be
used for screening of agents modulating subtype C LTR mediated gene
expression.
147
Table 5.1: HIV-1 reporter cell lines. Abbreviations of reporter genes:
CAT, Chloremphenicol acetyl transferase; β-gal, β-galactosidase; Luc,
Luciferase; SEAP, Secretory alkaline phosphatase; GFP, Green fluorescent
protein.
Reporter gene
Parent Cell line Reference
CAT CEM Merzouki et al, 1995
Hela-CD4 Akrigg et al, 1991, Kimpton et al, 1992
Hela-CD4-CXCR4 Vodicka et al, 1997 Hela-CD4-CCR5 Harrington et al, 2000 β-gal
P4 (Hela-CD4-LTR-β-gal) Kimpton et al, 1992, Charneau et al, 1992
1G5 (Jurkat - LTR-Luc) Aguilar-Cordova et al, 1994 Luc CEM-NKR-CCR5-LTR-Luc Spenlehauer et al, 2001 CEMX174-LTR-SEAP Means et al, 1997 SEAP MOLT4-SEAP-CCR5 Miyake et al, 2003 Hela-CD4-GFP Dorsky et al, 1996 PM1-CCR5-LTR-GFP Dorsky et al, 1999 CEM-GFP Gervaix et al, 1997 CEM-GFP-CCR5 Lelievre et al, 2004 GHOST-CCR5 (HOS-CD4-CCR5-LTR-GFP) Trkola et al, 1998
GFP
GHOST-CXCR4-LTR-GFP (HOS-CD4-CCR5-LTR-GFP) Trkola et al, 1998
β-gal-Luc JC53bl or TZMbl (Hela-CD4-CXCR4-CCR5-LTR-βgal-LTR-Luc)
Wei et al, 2002
5.2 Materials and methods
5.2.1 Plasmids pCR-TOPOII, a TA cloning vector was procured from Invitrogen, USA. The
vectors pCDNA 3.1, pEGFP-1 and pIRES2-EGFP were obtained from Clontech,
USA. The pGL3 basic vector was obtained from Promega, USA. The expression
vector for subtype C Tat (C31S Tat) was a kind gift of Dr. Ranga Uday Kumar,
JNCASR, India (Ranga et al, 2004). pIndie-C1, an infectious full length
molecular clone of HIV-1 subtype C Indian isolate was kindly provided by Dr.
M. Tatsumi, Japan (Mochizuki et al,1999). The subtype B Tat expression vector
148
pCDNA-Tat has been described previously from this laboratory (Joseph et al,
2003).
5.2.2 Construction of pLTRC-EGFP, pLTRC-Luc and pLTRC-
Luc-EGFP vectors The subtype C 5′ LTR spanning 592 bases of “U3-R-U5” region was amplified by
polymerase chain reaction (PCR) from pIndie-C1 molecular clone using forward
primer (5’TCACACACAAGGCTTCTTCC, 56 to 75 of Indie-C1 genome) and
reverse primer (5’CTGTTCGGGCGCCACTGCTA, 648 to 629 of Indie-C1
genome). The PCR parameters used was as follows: denaturation at 94 ˚C (1
min.), annealing at 55 ˚C (1 min.), extension at 72 ˚C (1 min.) for 35 cycles
followed by a 10 min. extension at 72 ˚C. PCR amplified subtype C LTR
fragment was first cloned into pCR-TOPOII (Invitrogen, USA) cloning vector
according to the manufacturer’s protocol and the resulting vector was named as
pCR-TOPOII-LTRC. The sequence of cloned LTR was confirmed by DNA
sequencing (ABI 310 Genetic Analyzer, ABI, USA).
5.2.2.1 pLTRC-EGFP HIV-1 subtype C LTR fragment was taken out from pCR-TOPOII-LTRC by
Xho1/BamH1 restriction digestion and was cloned in front of EGFP gene in Xho1
and BamH1 sites of pEGFP-1 vector. The resultant vector was named as pLTRC-
EGFP, a single reporter vector that has subtype C LTR promoter driving the
EGFP reporter gene.
5.2.2.2 pLTRC-Luc The Xho1/BamH1 restriction fragment of HIV-1 LTR from pCR-TOPOII-LTRC
was cloned in front of Luciferase gene in Xho1 and BglII sites of pGL3 Basic
vector. The resultant vector was named as pLTRC-Luc, a single reporter vector
that has subtype C LTR promoter driving the Luciferase reporter gene.
149
5.2.2.3 pLTRC-Luc-EGFP
We have used pIRES2-EGFP, a CMV promoter regulated vector having Internal
Ribosomal Entry Site (IRES) and Enhanced Green Fluorescence Protein (EGFP)
immediately after MCS for constructing the dual reporter vector. CMV promoter
was taken out from pIRES2-EGFP by digesting it with Ase1, which cuts at the 5’
end of CMV promoter and the digested vector was filled in and blunt ended with
Klenow fragment (Roche, Germany). It was then digested with Nhe1 enzyme to
remove CMV promoter. The LTRC-Luc fragment was taken out from pLTRC-
Luc mentioned above using Sma1/Xba1 double digestion and was cloned in to the
CMV promoter less pIRES2-EGFP vector at Sma1 and a compatible cohesive end
of generated Xba1 site. The resultant vector was named as pLTRC-Luc-EGFP, a
dual reporter vector that has subtype C LTR promoter driving both EGFP and
Luciferase reporter genes. The cloned LTR sequence in new reporter vectors were
reconfirmed by nucleotide sequencing. RT-PCR analysis of RNA isolated from
transfected cells was performed using following sets of GFP and Luc primers. For
GFP gene, DM472 (AgAACggCATCAAggTgAAC) forward primer and DM473
(gAACTCCAgCAggACCATgT) reverse primer were used. For Luc gene,
DM476 (CgCATgCCAgAgATCCTATT) forward primer and DM477
(AgCAgCgCACTTTgAATCTT) reverse primer were used.
5.2.3 Cell culture and transfections HEK293T cells obtained from NCCS cell repository were cultured in DMEM
medium supplemented with 10% Fetal Bovine Serum (Invitrogen, USA), 100
U/ml of penicillin and 100 μg/ml of Streptomycin in incubator maintaining 37 ˚C
temperature and 5% CO2. For transfection of plasmids, HEK293T cells were
seeded at a density of 5 Χ 105 cells/well in a six-well plate and incubated for 12 to
16 hours to obtain 60-70% confluence. Cells were supplemented with fresh
medium 4 hours before transfection and later 3-4 μg of plasmids were transfected
with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s
instructions. After 24 to 36 hours post transfection, cells were washed with 1 X
PBS and then cells were trypsinized and lysed in 100µl of 1X cell culture lysis
150
buffer (Promega, USA). Protein concentrations in cell lysates were measured
using Bio-Rad protein assay reagent (Bio-Rad, USA).
5.2.4 EGFP visualization and quantitation EGFP expression was initially visualized and monitored using an Olympus IX-70
Fluorescence microscope with filter for EGFP visualization. In order to get a
quantitative value of EGFP expression, transfected cells were fixed in 3.7% Para-
formaldehyde, washed with 1X PBS and were analyzed by flow cytometry using
FACS Vantage flow cytometer (Becton Dickinson, USA). The FL1 emission
channel was used to monitor EGFP expression. In the histogram of fluorescence
intensity of living cells, transfected cells that emitted a fluorescent signal above
background were distinguished from untransfected cell population. Since transient
transfection assay system shows varied levels of EGFP expressing heterogeneous
cell population, we have considered mean fluorescence intensity (MFI) of the
population of fluorescent cells for comparative analysis. MFI of transfected cells
were compared in the presence and absence of HIV-1 transactivator Tat gene. The
EGFP expression in transfected cell lysates was also quantitated as described
from our laboratory (Dandekar et al, 2005). In this, cells were washed with 1X
PBS and 125 μl of 1X cell culture lysis buffer (Promega, USA) was added to lyse
the cells and lysates were incubated on ice for 30 min. Then lysates were
centrifuged at 13,000 g for 10 min. and supernatants were collected and assayed
for the amount of protein using Bradford reagent (Biorad, USA). Then different
concentrations of protein were used to quantitate GFP by fluorimetry in
Fluoroscan Ascent FL (Thermo Labsystems, Finland).
5.2.5 Luciferase assays HEK293T cells transfected with LTR reporter gene constructs were harvested
after 24-36 hours post transfection, washed and then lysed as described in section
5.2.4. The lysates were used to quantitate luciferase activity using Luclite
Luminescence reporter gene assay system kit (Perkin-Elmer Life Sciences, USA)
according to the manufacturer’s protocol. Briefly, 100 µl of protein lysate in cell
151
culture lysis buffer was taken in a 96 well plate (Packard Biosciences, USA) and
100 µl of substrate was added in dark. Luminescence of samples was measured in
a Top Count microplate reader (Packard Biosciences, USA).
5.3 Results Recent HIV-1 prevalence data clearly indicate that there has been a shift in the
prevalence from B to C subtype in last few years due to the pandemic in Africa
and Asia (Spira et al, 2003). Currently more than 50% infection worldwide is
believed to be with subtype C viral isolates. As majority of earlier work has been
performed with subtype B isolates/sequences, the literature about subtype C
remains relatively scanty. Several studies have indicated that subtypes B and C
differ markedly in their properties (Naghavi et al, 1999; Spira et al, 2003) and
hence it becomes more important to study subtype C in further detail.
5.3.1 Analysis of GFP and Luciferase expression in cells
transfected with reporter vectors and Tat The transcriptional activity of LTRC-reporter vectors has been analyzed by
transfection of individual plasmids in HEK293T cells and expression of EGFP
and Luciferase enzymatic activity was measured in transfected cell lysates. The
EGFP expression was initially visualized by fluorescence microscopy and flow
cytometry, however; further quantitative analysis was performed using
fluorimetry of transfected cell lysates. The Luciferase activity was measured by
commercially available enzymatic assay described in section 5.2.5. The basal
level of EGFP fluorescence was observed to be more in pLTRC-EGFP as
compared to the dual reporter vector pLTRC-Luc-EGFP as visualized by
fluorescence microscopy (Figure-5.2A). Mean fluorescence intensity (MFI)
derived from flow cytometry of transfected 293T cells show that basal EGFP
fluorescence was at least two fold lower in dual reporter vector pLTRC-Luc-
EGFP as compared to pLTRC-EGFP (Figure-5.2B). To analyse the differences in
EGFP and Luciferase expression from pLTRC-Luc-EGFP reporter in the absence
and presence of Tat, EGFP and Luciferase RNA levels were quantitated in dual
152
reporter and subtype C Tat co-transfected cells. Low level of EGFP expression
was observed as compared to the Luciefrase (Figure-5.3). GAPDH was used as
control.This type of difference in level of basal EGFP expression under the
Fig 5.1: Schematic representation of construction of pLTRC-Luc, pLTRC-
EGFP and pLTRC-Luc-EGFP vectors.
control of LTR promoter could be attributed to the presence of IRES sequence
before EGFP open reading frame in dual reporter (Mijakovic et al, 2005). Co-
transfection of HIV-1 transactivator protein Tat, which is very important for
initiation and elongation of LTR mediated transcription, clearly induces the
pIndie-C1 (Subtyep C)
P lac
lacZ
f1 ori
KanAmp
pUCori A
MCS
pCR II TOPO-LTRC pLTRC-Luc
Luc
SV40 Poly Aori
Amp
f1 oriPoly A
MCS
B
pLTRC-EGFP
EGFPSV40 Poly A
f1 oriP
oriP
Kan
HSVTKPoly A
oriMCS
C
pLTRC-Luc-EGFP
IRES
EGFP
MCSoriHSV TK Poly A
KanoriP Pf1ori
SV40Poly A
D
PCR amplification
LTRC
LTRC
LTRC
LTRC
LTRC Luc
LTRC LTRCpIndie-C1 (Subtyep C)
P lac
lacZ
f1 ori
KanAmp
pUCori A
MCS
pCR II TOPO-LTRC pLTRC-Luc
Luc
SV40 Poly Aori
Amp
f1 oriPoly A
MCS
B
pLTRC-EGFP
EGFPSV40 Poly A
f1 oriP
oriP
Kan
HSVTKPoly A
oriMCS
C
pLTRC-Luc-EGFP
IRES
EGFP
MCSoriHSV TK Poly A
KanoriP Pf1ori
SV40Poly A
D
PCR amplification
LTRC
LTRCLTRC
LTRC
LTRC
LTRC Luc
LTRC LTRC
153
Fig 5.2: EGFP expression in 293T cells transfected with LTRC-reporter vectors
and transactivation by HIV-1 subtype C Tat.
A. Bright Field (BF) and fluorescence microscopic images under ultraviolet
(FL) light of 293T cells transfected with i) pLTRC-EGFP without Tat; ii)
pLTRC-EGFP with Tat; iii) pLTRC-Luc-EGFP without Tat; iv) pLTRC-Luc-
EGFP with Tat.
B. Analysis of EGFP fluorescence of 293T cells transfected with LTRC-reporter
vectors in the presence and absence of subtype C Tat by flow cytometry. The
values in the histogram indicate mean fluorescence intensity.
Fig 5.3: Expression of Luciferase and EGFP in pLTRC-Luc-EGFP and
pCDNA or pTatC transfected 293T cells by reverse transcription-polymerase
chain reaction (RT-PCR).
LUC
EGFP
GAPDH
pLTRC-Luc-EGFP (µg) 1 1 2 2 3 3pCDNA (1µg) + - + - + -pTat C (1µg) - + - + - +
LUC
EGFP
GAPDH
LUC
EGFP
GAPDH
pLTRC-Luc-EGFP (µg) 1 1 2 2 3 3pCDNA (1µg) + - + - + -pTat C (1µg) - + - + - +
48 30
231 1434
59 652
B +pCDNA +pTAT C
pLTRC-Luc
pLTRC-EGFP
pLTRC-Luc-EGFP
i
ii
iii
iv
BF FLA
48 30
231 1434
59 652
B +pCDNA +pTAT C
pLTRC-Luc
pLTRC-EGFP
pLTRC-Luc-EGFP
i
ii
iii
iv
BF FLA
154
Fig 5.4: Transcriptional activity of subtype C reporter vectors in presence and
absence of subtype B (pTAT B) and C Tat (pTAT C).
A. Measurement of EGFP expression in 293T cell lysates transfected with
different LTRC-reporter vectors in the presence and absence of subtype B and
C Tats.
B. Measurement of Luciferase enzymatic activity in 293T cell lysates
transfected with different LTRC-reporter vectors in the presence and absence of
subtype B and C Tats.
expression of both EGFP and Luciferase. We have co-transfected subtype C Tat
expressing vector C31S Tat (Ranga et al, 2004) along with LTRC-reporter vectors
to study transactivation. As expected subtype C Tat was able to
pLTRC-LucpLTRC-EGFP pLTRC-Luc-EGFP pCDNApTAT B pTAT C
GFP
uni
ts
+ + + - - - - - -- - - + + + - - -- - - - - - + + ++ - - + - - + - -- + - - + - - + -- - + - - + - - +
0
A
20
40
60
B
Luci
fera
se u
nits
20000
40000
pLTRC-LucpLTRC-EGFP pLTRC-Luc-EGFP pCDNApTAT B pTAT C
+ + + - - - - - -- - - + + + - - -- - - - - - + + ++ - - + - - + - -- + - - + - - + -- - + - - + - - +
0
10000
30000
50000
pLTRC-LucpLTRC-EGFP pLTRC-Luc-EGFP pCDNApTAT B pTAT C
GFP
uni
ts
+ + + - - - - - -- - - + + + - - -- - - - - - + + ++ - - + - - + - -- + - - + - - + -- - + - - + - - +
0
A
20
40
60
B
Luci
fera
se u
nits
20000
40000
pLTRC-LucpLTRC-EGFP pLTRC-Luc-EGFP pCDNApTAT B pTAT C
+ + + - - - - - -- - - + + + - - -- - - - - - + + ++ - - + - - + - -- + - - + - - + -- - + - - + - - +
0
10000
30000
50000
155
transactivateexpression of reporter genes with all the three reporter vectors.
Enhancement of EGFP expression due to Tat was visualized by fluorescence
microscopy (Figure-5.2A) for both pLTRC-EGFP and dual reporter vector
pLTRC-Luc-EGFP. About 3-fold increase in mean fluorescence intensity (MFI)
of EGFP expressing cells was observed with both the reporter vectors (Figure-
5.2B). pLTRC-Luc vector alone or with Tat did not show any EGFP expression
and served as a negative control. We further quantitated EGFP expression in
transfected cell lysates by fluorimetry as previously described (Dandekar et al,
2005) and described in section 5.2.4. Fluorimetric quantitation of transfected cell
lysates indicated several fold increase in EGFP expression in presence of subtype
C Tat as compared to the basal levels. Subtype C Tat increased EGFP expression
to about 12 fold in pLTRC-Luc-EGFP whereas transactivation was seen to be 22
fold in case of pLTRC-EGFP vector (Figure-5.4A). Then we measured Luciferase
activity for reporter vectors in presence and absence of co-transfected Tat.
Analysis of basal Luciferase expression in both pLTRC-Luc and the dual reporter
vector shows similar low level of activity (Figure 5.4B). Around 46-fold increase
in Luciferase activity was seen in case of cells transfected with Tat and pLTRC-
Luc-EGFP whereas 55-fold transactivation was observed with pLTRC-Luc
(Figure-5.4B). Increase in fold transactivation observed by Luciferase activity
could be due to high sensitivity of the Luciferase reporter gene assay. Further
more to test whether the subtype C LTR based reporter vectors can also be
transactivated by Tat from subtype B, we have co-transfected pCDNA-Tat
(Joseph et al, 2003) expressing subtype B Tat. Transactivation was observed in all
the three vectors in presence of subtype B Tat (Figure 5.4). Thus the constructed
subtype C vectors can function efficiently as reporter vectors.
5.3.2 Analysis of GFP and Luciferase expression in cells
transfected with reporter vectors and subtype C molecular clone As stated earlier, co-transfection of HIV-1 molecular clones along with LTR-
reporter vector or viral infection of a cell line having stably integrated LTR-
reporter gene provides a sensitive and quantitative measure of virus infectivity
156
Fig 5.5: Transcriptional activity of the single and dual reporter vectors upon co-
transfection of subtype C molecular clone pIndie-C1 in 293T cells.
A. EGFP expression in transfected 293T cells in presence of pIndie-C1 virus.
B. Luciferase activity of transfected 293T cells in presence of pIndie-C1 virus.
and replication. Tat protein expressed by virus leads to transactivation of LTR
regulated reporter gene. In order to test the reporter gene expression in presence
of HIV-1, we transfected single and dual reporter vectors along with subtype C
HIV-1 molecular clone, pIndie-C1. After 36 hours post transfection, EGFP and
Luciferase activity was detected in lysates of transfected 293T cells, which clearly
showed around 6 to 7 fold increase in EGFP and 12 to 15 fold increase in
Luciferase expression respectively in the presence of pIndie-C1 (Figure-5.5A and
B). This data clearly indicate that the single reporter vectors and dual reporter
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
0
10
20
30
40
50
GFP
uni
ts
A
B
Luci
fera
se u
nits
5000
10000
15000
20000
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
0
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
0
10
20
30
40
50
GFP
uni
ts
A
B
Luci
fera
se u
nits
5000
10000
15000
20000
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
0
B
Luci
fera
se u
nits
5000
10000
15000
20000
pLTRC-EGFPpLTRC-LucpLTRC-Luc-EGFP pCDNA pIndie-C1
+--+-
+---+
-+-+-
-+--+
--++-
--+-+
0
157
vectors constructed in the present study can be used not only for studying the
subtype C LTR but also for analysis of viral gene expression.
5.3.3 Sensitivity of GFP and Luciferase detection from the dual
reporter vector Among all the reporters used till date, Luciferase has been widely used for
Fig 5.6: Comparison of gene expression from EGFP and Luciferase reporter
gene in the dual reporter vector at different concentrations of transfected cell
lysate.
A) Fold transactivation of EGFP and Luc expression from the dual vector at
different concentration of cell lysate when co-transfected with subtype C Tat
expression vector.
B) Fold transactivation of EGFP and Luc expression from the dual vector at
different concentration of cell lysate when co-transfected with subtype C
molecular clone pIndie-C1.
0
10
20
30
B
EGFPLuc
0
10
20
30
40
50
A
EGFPLuc
Conc. of protein (µg) 10010 505
Conc. of protein (µg) 10010 505
Fold
tran
sact
ivat
ion
by p
TatC
Fold
tran
sact
ivat
ion
by p
Indi
e-C
1
0
10
20
30
B
EGFPLuc
0
10
20
30
40
50
A
EGFPLuc
Conc. of protein (µg) 10010 505
Conc. of protein (µg) 10010 505
Fold
tran
sact
ivat
ion
by p
TatC
Fold
tran
sact
ivat
ion
by p
Indi
e-C
1
158
quantitation of gene expression due to its high sensitivity and ease of assay.
pIndie-C1 transfected cells were checked for both EGFP and Luciferase
expression at various concentration of lysate protein starting from 5 to 100 μg. In
subtype C Tat as well as pIndie-C1 co-transfected cells, Luciferase activity
showed consistently more fold increase than EGFP reporter expression at all
concentrations of protein (Figure- 5.6A and 5.6B). More or less similar fold
increase in Luciferase activity was observed at all the concentrations of protein
tested in the assay, where as EGFP fluorescence showed smaller increase at lower
concentrations, however, at higher protein concentrations, similar increase in
EGFP expression was observed (Figure-5.6A and 5.6B). This clearly indicates
that even though EGFP reporter gene quantification assay is less sensitive as
compared to Luciferase activity assay, it can still be used at higher concentrations
of protein for quantitative analysis of LTR-directed transcriptional activity.
5.3.4 Screening the effect of transcription modulators on viral
LTR promoter using dual reporter vector The effect of viral transcription modulators can be evaluated by reporter gene
assay in which an LTR-reporter vector is co-transfected along with the Tat
expressing plasmid followed by treatment with viral transcription modulator and
analysis of reporter gene expression. pLTRC-Luc-EGFP was co-transfected with
pIndie-C1 molecular clone and treated with a nucleoside analogue 5,6-dichloro-1-
beta-D-ribofuranosylbenzimidazole (DRB), which inhibits Tat-associated kinase
activity and thereby LTR transcription (West et al, 1999) or Sodium Butyrate, a
histone deacetylase inhibitor that markedly induces HIV-1 transcriptional activity
(Van Lint et al, 2000). DRB inhibited LTRC dependent EGFP and Luciferase
expression, whereas Sodium Butyrate increased the reporter gene expression
(Figure-5.7A and 5.7B). Furthermore, as expected inhibition of LTR mediated
transcription by DRB treatment led to decrease in virus production whereas
Sodium Butyrate increased the virus production (Figure-5.7C). Thus dual reporter
159
Fig 5.7: Effect of transcription modulators on LTR promoter in pIndie-C1 and
pLTRC-Luc-EGFP co-transfected 293T cells: A) Analysis of EGFP expression
in cell lysates. B) Analysis of Luciferase expression in cell lysates and C) Virus
released into the culture supernatant as quantified using HIV-1 p24 antigen
enzyme-linked immunosorbent assay. UT, untreated; DRB, 5,6-dichloro-1-D-
ribofuranosylbenzimidazole; NaB, sodium butyrate.
vector constructed in the present study will also be useful for screening of viral
transcription modulators.
5.4 Discussion HIV-1 LTR-reporter gene constructs have been widely used not only to
understand the LTR promoter activity but also to identify specific DNA
sequences in the promoter which are bound by viral and host proteins that
regulate viral gene expression (Roebuck et al, 1999; Pereira et al, 2000). Most of
the work on viral gene expression till date has been carried out using subtype B
LTR. Recent comparison of subtypes indicates significant differences between
subtype B and C viral isolates and thus necessitates further detailed
C
0
0.25
0.50
0.75
1.00P2
4 in
ng/m
l
UT DRB NaB
0
10
20
30
Fold
tran
sact
ivat
ion
(EG
FP)
A
UT DRB NaB
B
0
20
40
60
80
Fold
tran
sact
ivat
ion
(Luc
)
UT DRB NaB
C
0
0.25
0.50
0.75
1.00P2
4 in
ng/m
l
UT DRB NaB
0
10
20
30
Fold
tran
sact
ivat
ion
(EG
FP)
A
UT DRB NaB0
10
20
30
Fold
tran
sact
ivat
ion
(EG
FP)
A
UT DRB NaB
B
0
20
40
60
80
Fold
tran
sact
ivat
ion
(Luc
)
UT DRB NaB
160
characterization of subtype C viruses (Spira et al., 2003). Current prevalence of
HIV-1 subtype C viral infections worldwide and relatively little literature about
the biology of subtype C virus, demands further study of this subtype and
development of reagents useful for research. The first report of a subtype C LTR
reporter vector came in a study by (Naghavi et al, 1999), in which LTRC-CAT
vector was made using most of the U3-R region (position –382 to +113) and was
used to compare subtype C LTR promoter activity with A, B, D, E and G
subtypes. Their results indicated highest transcriptional activity in subtype C
LTR, which was thought to be due the presence of a third NFκB binding site
(Naghavi et al., 1999). In a later study, subtype C LTR-Luc vector was used in
comparative analysis of LTR of different subtypes, which showed that subtype E
was most potent LTR (Jeeninga et al., 2000). The subtype C LTR-Luc vector
used in that study had only the LTR spanning basal promoter, enhancer and TAR
region (position –147 to +63) but excluded the modulatory region (position –340
to –184). They later also showed that replication rates could vary between
different viral subtypes due to host cell environment (van Opijnen et al., 2004).
Transcription of HIV-1 proviral genome is regulated by a combinational effect of
viral proteins and cellular transcription factors that interact with specific LTR
sequences ranging from TAR region to the modulatory region. In this study, we
have used LTR sequences spanning all four functional regulatory regions of HIV-
1 transcription, i.e., transactivation response (TAR) element, core promoter, core
enhancer and most of the modulatory region (position –399 to +193) from Indian
subtype C HIV-1 infectious molecular clone pIndie-C1 (Mochizuki et al, 1999)
and established a subtype C LTR regulated dual reporter vector pLTRC-Luc-
EGFP. In this novel vector, the subtype C LTR promoter controls the expression
of both EGFP and Luciferase reporter genes, which provides a useful system to
study subtype C promoter activity. This dual reporter vector has many advantages
over the single reporter vectors. EGFP reporter gene expression can easily be
detected in live cells by fluorescence without the need of cell lysis, fixation or
additional substrate for assay and is an excellent marker for visualization of
molecular and cellular events in transformed cell lines (Yu et al, 2003). Due to
161
the expression of both EGFP and Luciferase, this dual reporter can be used for
normalization of transfection efficiency without co-transfection of a reference
reporter plasmid. EGFP expression can also be used for sorting of cells and easy
visualization where as Luciferase is the most widely used reporter gene for
quantitative analysis of promoter activity because of its high sensitivity as
compared to CAT, ß galactosidase, SEAP, etc (Arnone et al, 2004). However,
EGFP expression was little less as compared to the Luciferase expression in dual
reporter vector and Tat expressing plasmid co-transfections which can be
attributed to the presence of EGFP gene after IRES sequences in dual reporter
vector but this does not affect the functionality of the vector (Mijakovik et al,
2005). Use of this dual reporter vector allows one to study promoter activity by
various methods such as fluorescence microscopy, flow cytometry and
fluorimetry for EGFP expression and luminometry for quantitation of Luciferase
activity. Moreover quantitation of EGFP expression by fluorimetry provides an
alternative quantitative method for reporter studies in resource poor settings. The
effect of transcriptional modulators on viral transcription can be evaluated with
the constructed subtype C LTRC-reporter vectors. The reporter vector is co-
transfected with Tat and thereafter treated with transcription modulators to
observe their effect on LTR directed reporter gene activity. The DRB, a
transcriptional inhibitor and Sodium butyrate, a transcriptional activator was used
to evaluate their effect. Reduction in LTR directed reporter gene expression was
observed with DRB treatment whereas Sodium butyrate increased the reporter
gene activities from the dual reporter vector. Thus the constructed reporter vectors
are also useful for screening of viral transcription modulators. Finally, this novel
subtype C reporter vectors can be used both for easy visualization and sensitive
quantitation of LTR mediated gene expression and thus will become a useful tool
for HIV research.
162
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