(Received for publication, September 19, 1996, and in revised form, February 25, 1997)
From the UCLA Department of Medicine, Division of
Hematology-Oncology, UCLA AIDS Institute, Los Angeles, California
90095, the
Children's Hospital Los Angeles, University of
Southern California School of Medicine, Los Angeles, California
90027, and the ¶¶ University of Rochester Cancer Center and
Departments of § Medicine, ¶ Microbiology, ¶ Immunology, and
** Biochemistry, University of Rochester, Rochester, New York 14642
Cellular tRNALys-3 serves as the primer for reverse transcription of human immunodeficiency virus, type 1 (HIV-1). tRNALys-3 interacts directly with HIV-1 reverse transcriptase, is packaged into viral particles and anneals to the primer-binding site (PBS) of the HIV-1 genome to initiate reverse transcription. Therefore, the priming step of reverse transcription is a potential target for antiviral strategies. We have developed a mutant tRNALys-3 derivative with mutations in the PBS-binding region such that priming specificity was re-directed to the highly conserved TAR stem-loop region. This mutant tRNA retains high-affinity binding to HIV-1 reverse transcriptase, viral encapsidation, and is able to prime at both the targeted TAR sequence and at the viral PBS. Constitutive expression of mutant tRNA in T-cells results in marked inhibition of HIV-1 replication, as determined by measurements of viral infectivity, syncytium formation, and p24 production. Inhibition of retroviral replication through interference with the normal process of priming constitutes a new anti-retroviral approach and also provides a novel tool for dissecting molecular aspects of priming.
Retroviruses contain two copies of an RNA genome which replicates via a DNA intermediate (1). Transcription of the RNA genome into the DNA intermediate is performed by the viral enzyme reverse transcriptase (RT).1 The primer for reverse transcription of the retroviral genome is a cellular transfer RNA (tRNA). tRNA is also crucial in several steps of the reverse transcription process subsequent to priming, such as the strand-transfer reactions. Human immunodeficiency viruses (HIV) and their simian counterparts utilize tRNALys-3 as their primer (2, 3). HIV-1 virions appear to contain approximately eight tRNALys-3 molecules per two copies of viral genome (4).
The reverse transcriptase protein of HIV-1 has been shown to directly
bind the tRNALys-3 primer in vitro (5-8). The
interaction between tRNALys-3 and HIV-1 reverse
transcriptase is complex. At least four cellular tRNAs have been
isolated from HIV-1 virions including tRNALys-3,
tRNALys-1, tRNALys-2, and tRNAIle
although tRNALys-3 is the predominant species isolated (3).
Several interactions have been demonstrated in vitro between
HIV-1 RT and tRNALys-3. Barat et al. (5) have
demonstrated specific contacts with the anticodon loop. Nuclease
footprinting suggests partial protection of the so-called D-loop, TC
loop, and anticodon loops by RT (6).
Mutations in the TC loop, and particularly the D-loop of
tRNALys-3 were found to significantly impair binding to
HIV-1 RT in gel shift assays (8). Consensus RNA motifs selected
in vitro by the SELEX procedure demonstrate that a consensus
RNA pseudoknot with significant secondary and tertiary homology to the
D-loop of tRNALys-3 is a preferred binding motif to RT (9).
Additionally, RT facilitates annealing of the tRNALys-3 to
the PBS sequence in vitro suggesting RT also plays a role in
unwinding of the tRNALys-3 acceptor stem (8). Finally,
while RT-deficient virions contain a viral genome, the
tRNALys-3 primer is not packaged, indicating that
tRNALys-3 packaging is dependent upon the presence of RT
(8). In addition to the primary tRNA-RT interaction required for
packaging, RT also recognizes preformed tRNA-HIV-1 template complexes
in vitro suggesting a second level of interaction likely to
be important in initiation of cDNA synthesis (8).
Selective incorporation of tRNALys-3 is thought to be
mediated by the Pr160gag-pol precursor. Precursor
processing is not required for selective incorporation (3).
Incorporation is also not dependent on the primer-binding site.
Interactions have been described between the p66 as well as the p51
subunits of RT and the 5 end of tRNA by platinum cross-linking (10).
Preincubation of heterodimeric RT (p66/p51) was noted to increase
catalytic activity on a poly(A)-oligo(dT) template (11) suggesting that
tRNA binding may also lead to "activation" of the p66/p51
heterodimer. Nuclease footprinting of HIV-1 RT-tRNALys-3
complexes demonstrated protection of bases within the anticodon and
D-loops, and potential T
C-D-loop interactions which are disrupted in
the presence of HIV-1 RT (6). Other investigators, using pancreatic
RNase A footprinting, showed protection of both the tRNALys-3 anticodon loop and D-loop but not the acceptor
stem, confirming a strong HIV-1 RT interaction with several regions of
tRNA (7).
In addition to interaction between the tRNA primer and HIV-1 RT, other
interactions involving the tRNA primer include annealing of the
3-terminal 18 base pairs of tRNALys-3 homologous to the
HIV-1 PBS and annealing of various loops of tRNALys-3 to
regions of the HIV-1 genome outside the PBS (12-18). HIV-1 particles
containing viral RNA sequences lacking the PBS continue to
preferentially package tRNALys-3, suggesting that PBS-tRNA
annealing alone does not determine the packaging preference for the
tRNALys-3 primer (3).
Anti-retroviral strategies which make use of the unique properties of tRNAs have recently been proposed. Rossi and co-workers (19) described a chimeric tRNA in which anti-HIV-1 ribozyme was fused to tRNALys-3. Because this chimeric RNA contained tRNALys-3 coding sequences, it was expressed at high levels via RNA polymerase III. In addition, this ribozyme/tRNA hybrid was specifically targeted to HIV-1 virions based on the affinity of tRNALys-3 for RT. Another study described a chimeric tRNA in which an anti-HIV-1 antisense RNA was linked to tRNAPro (20). In that case, the presence of tRNAPro sequences increased stability of the chimeric RNA in the intracellular environment (20).
We have developed a strategy for inhibition of the HIV-1 replication cycle via interference with priming of reverse transcription. To accomplish this, we designed a tRNALys-3-derived mutant tRNA with a modified acceptor stem. This mutant tRNA, designated tRNAtarD, was shown to inhibit HIV-1 replication when expressed in human CD4 lymphocytes. The results presented here, together with available published information, indicate that potent inhibition of retroviral replication can be achieved by targeting priming of reverse transcription.
Inhibition of retroviral replication through interference with the normal process of priming constitutes a novel anti-retroviral approach and also provides a powerful tool for dissecting molecular aspects of priming.
The tRNALys-3-UUU gene (21)
with 5- and 3
-flanking sequences was obtained by amplification of DNA
extracted from HeLa cells using the primers: 5
-
TCGCCGAGATAAGCTTCAGCCTCTACTATGGTACAG-3
and
5
-ATAATAGCACAAGCTTTATTACCCTCCACCGTCGTT-3
and Vent DNA polymerase (New
England Biolabs) according to the manufacturer's instructions. The PCR
fragment was then digested with HindIII and cloned into the
HindIII site of pBluescript-KS vector, to yield
pM13Lys-3. The tRNAtarD gene, including 20 nucleotides
upstream and 130 nucleotides downstream flanking the human
tRNALys-3-UUU gene, was obtained by PCR amplification using
two primer pairs: 1L,
5
-GTAAAGCTCTCGTGAAGATAGACCATAGCTCAGTCGGTAGAGC-3
and 1R,
5
-CCAAAAGCAAAGACATGCCGCTTAGACCACAGGGACTTGAACCCTGGAC-3
; and 2L,
5
-GTCCAGGGTTCAAGTCCCTGTGGTCTAAGCGGCATGTCTTTGCTTTTGG-3
and 2R, 5
-ATAATAGCACAAGCTTTATTACCCTCCACCGTCGTT-3
. The
first amplification was done using the pM13Lys-3 DNA
template and primers 1L and 1R, resulting in fragment A. A second
amplification was carried out using pM13Lys-3 and primers
2L and 2R, giving rise to fragment B. Both A and B fragments were then
agarose-gel purified. A third PCR reaction was performed using a
mixture of purified A and B fragments as templates. Primers 1L and 2R
were used to generate a tRNAtarD sequence containing 5
- and
3
-flanking sequences required for correct transcription and processing
by polymerase III. The final PCR fragment was cloned into the blunted
XhoI site of pAC7, an AAV-derived vector (22), resulting in
pACtarD. The sequence was then verified. tRNAtarD coding sequences were
excised from pACtarD by HindIII and NcoI
digestion. After complete filling of the ends formed by
HindIII and NcoI, the fragment was subcloned into
the SnaBI site of the vector pN2A (23), resulting in the retroviral vector pN2A-tarD (Fig. 1C), and sequence and
orientation were confirmed by sequencing.
The vectors pUClys and pUCtarD contain coding sequences for both
tRNALys-3 and tRNAtarD downstream of the T7 promoter and
were used for in vitro transcription. Coding sequences for
tRNALys-3 were synthesized by annealing and extending two
oligonucleotides: left primer
5-AATTGCTGCAGTAATACGACTCACTATAGCCCGGATAGCTCAGTCGGTAGAGCATCAGACTTTTAATCTGAGGGTCCAGGGTTCAAG-3
; and right primer
5
-CTAAGCTGCAGATGCATGGCGCCCGAACAGGGACTTGAACCCTGGACCCT-3
at 70 °C
for 15 min followed by 37 °C for 30 min in 1 × PCR buffer using Vent polymerase (New England Biolabs). The resulting PCR fragment
was digested with PstI and cloned into the PstI
site of pUC120 to yield pUClys. The tRNAtarD sequences were generated by amplification of pUClys using the primer pair: left
5
-AATTGCTGCAGTAATACGACTCACTATATAGACCATAGCTCAGTCGGTAGAGCA and
right 5
-CTAAGCTGCAGATGCATGGTTAGACCACAGGGACTTGAACCCTGGACCCT. The
resultant PCR fragment was then cloned into the SmaI site of
pUC18 to yield pUCtarD.
The vector pSP-PBS was constructed by subcloning a 0.9-kilobase pair,
ScaI to NsiI fragment from the HIV-1 molecular
clone pNL4-3 (24), into the bacterial vector, pSP-73 (Promega Corp., Madison, WI), previously digested with PvuII and
NsiI. The restriction fragment from pNL4-3 includes
nucleotides 133 to +386 of the flanking sequences and 5
-LTR region
of HIV-1-NL4-3 (24).
pUClys and pUCtarD were linearized by
NsiI digestion to generate a 3 CCA end, and transcribed by
T7 polymerase using reagents and conditions supplied with the Promega
T7 transcription kit in the presence of 10 µCi of
[32P]CTP. Radiolabeled RNAs were gel purified according
to the method described previously (25). Competition analysis was
performed by standard methods (26).
The GP+E-86 ecotropic packaging cell line was a generous gift from A. Bank (Columbia University, New York). The PA317 amphotropic packaging cell line and NIH3T3 fibroblasts were obtained from the American Type Culture Collection. The T-cell line MT-2 was obtained from the AIDS Research and Reference Reagent Program of the National Institutes of Health (Rockville, MD). The vector pN2A-tarD was transfected into the ecotropic packaging cell line GP+E-86 using lipofection (Life Technologies, Inc.). Transfected cells were selected in 0.5 mg/ml G418 (Geneticin, Life Technologies, Inc.). Virus-containing supernatant from the GP+E-86 cells was collected and used to transduce PA317 amphotropic packaging cells, and the cells selected in 0.5 mg/ml G418. High-titer clones were identified by measuring the transfer of G418 resistance using serially diluted supernatant to NIH3T3 fibroblasts, and supernatant from these clones was used to transduce MT-2 cells and CEM cells (ATCC).
The control vector, pLN, a MoMuLV-based retroviral vector containing the neomycin resistance gene expressed from the MoMuLV-LTR, was constructed and packaged in the laboratory of A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, WA).
To transduce MT-2 cells, LN and N2A-tarD vector-producing PA317 cells were irradiated at 40 gray and plated at 2 × 106 cells/100-cm2 plate 24 h before the addition of 1 × 106 MT-2 cells. Co-cultivation was carried out in the presence of 8 µg/ml Polybrene for 48 h. Non-adherent MT-2 cells were collected, and a second round of co-cultivation over irradiated vector-producing fibroblasts was performed. Transduced MT-2 cells were selected in 0.5 mg/ml G418, and subcloned by limiting dilution in 96-well plates, to yield MT2/LN (transduced with LN) and MT2/TARD (transduced with N2A-tarD) clones.
RT-PCR for Assay of tRNAtarD ExpressionTotal RNA was
extracted from parental MT2 and MT2/TARD cells using the acid
guanidinium isothiocyanate/phenol-chloroform method (27). The reverse
transcriptase reaction was carried out using MoMuLV reverse
transcriptase (Life Technologies, Inc.) following the manufacturer's
recommendations for first strand synthesis, using 100 ng of the
antisense primers described below. One µl of reverse transcribed
cDNA was amplified using 1 µl of RT reaction product as template.
Each PCR reaction was conducted in a final volume of 50 µl containing
1.25 unit of Taq DNA polymerase and a total of 300 ng of
tRNAtarD-specific primers (sense, 5-TAGACCATAGCTCAGTCGGT-3
; and
antisense, 5
-TGGTTAGACCACAGGGACTT-3
) or 300 ng of
tRNALys-3 specific primers (sense,
5
-GCCCGGATAGCTCAGTCGGT-3
; and antisense, 5
-TGGCGCCCGAACAGGGACTT-3
).
Thermocycling temperatures used were: initial denaturation at 94 °C
for 5 min, annealing at 60 °C for 2 min, and extension at 72 °C
for 2 min, followed by cycling at 94 °C for 1 min, 60 °C for 2 min, and 72 °C for 2 min for a total of 30 cycles. 10 µl of the
amplified PCR product was subjected to electrophoresis in a 2% agarose
gel.
Parental MT-2, MT2/LN cells which were transfected with retroviral vector alone (no tRNAtarD expression), and MT2/TARD were compared for ability to support HIV-1 replication. Cells were routinely grown in Iscove's medium (Life Technologies, Inc.) containing 10% fetal bovine serum and 1 × of Penn/Strep at 37 °C. The virus strain used in this study was HIV-IIIB, which was prepared and titered in MT-2 cells.
TCID50 AssayCells were seeded into 96-well plates at 5 × 103 cells/well. HIV-IIIB virus stock was serially diluted 10-fold in Iscove's medium containing no fetal bovine serum. Using 4-6 wells per dilution for each cell line, 0.1 ml/well of each virus dilution was inoculated. Control wells received the same amount of medium. Infected cultures were examined daily for syncytia formation and viral titers determined at days 5 and 10 postinfection according to the method described by Reed and Muench (28).
Infections and Viral p24 AssayFor each infection, a total of 1 × 104 cells (MT-2, MT2/TARD, or MT2/LN) in exponential growth phase were harvested, washed once with medium, and pelleted. The cell pellet was then resuspended in 1 ml of diluted HIV-IIIB stock containing 10 TCID50 units of virus. After adsorption at 37 °C for 2 h, 10 ml of medium was added, and the cells pelleted by centrifugation, resuspended in 15 ml of Iscove's and 10% fetal calf serum medium, and transferred into a 25-cm2 flask. Duplicate infections per cell line were employed in each challenge assay, and the infected cultures were incubated at 37 °C. Every other day beginning from day 2 postinfection, 0.5 ml of culture supernatant was removed from the flasks, and virus replication was monitored by measuring the production of p24 viral antigen in culture supernatant using an HIV-1 p24 antigen capture enzyme-linked immunosorbent assay (Coulter Immunology, Hialeah, FL).
RNA-dependent DNA Polymerase AssaysTemplate
RNAs comprising HIV-1 genome sequences for RNA-dependent
DNA polymerase assays in vitro were generated by in
vitro transcription from the plasmid clone, pSP-PBS (see "Vector
Construction" above). Template RNA 1, 545 nucleotides long, was
generated by transcription in vitro of an XmnI
restriction fragment pSP-PBS using T7 RNA polymerase. Template RNA 1 encompassed nucleotides 133 to +386 of the flanking sequences and
5
-LTR region of pNL4-3 (24). In addition, its 5
end contained 26 nucleotides from the multiple cloning site of pSP-73. Template RNA 2 was generated following restriction digestion of the plasmid pSP-PBS
with HindIII followed by transcription in vitro
using T7 RNA polymerase. Template RNA 2 contained the region of the
genomic RNA from
133 to +81, 240 nucleotides in length. This sequence
does not include the PBS. DNA primers 1, 2, and 3 were complementary to
regions of the template RNAs corresponding to HIV-1 genomic positions
+191 to +209, +59 to +78, and +22 to +41, respectively. Conditions for
annealing to and DNA polymerization from RNA templates with various
primers were as described previously (29). The RNA template and the
respective primers were annealed in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 80 mM KCl with a 10:1
molar ratio of primer to template. Similarly, RNA-dependent
DNA polymerase assays were carried out as before with slight
modifications (29). Final reaction mixtures contained 50 mM
Tris-HCl (pH 8.0), 1 mM dithiothreitol, 1.0 mM
EDTA, 34 mM KCl, 6 mM MgCl2, 2 nM substrate, 50 mM unlabeled dNTPs, and 4 units of recombinant purified HIV-1 RT. Recombinant HIV-1 RT was
graciously provided to us by Genetics Institute (Cambridge, MA). This
RT has native heterodimeric structure, is free of detectable nuclease
and polymerase contaminants, and has a specific activity of 40,000 units/mg (29). The RT was preincubated with the substrate for 5 min at
37 °C. The reaction was initiated by the addition of
MgCl2 and dNTPs, incubated for 15 min and terminated with
25 ml of termination mixture (90% formamide (v/v), 10 mM
EDTA (pH 8.0), and 0.1% each of xylene cyanol and bromphenol blue).
For some experiments, DNA synthesis was performed in the presence of
[
-32P]dCTP. When radiolabeled tRNA primers were
employed, unlabeled dNTPs were used in the synthesis reaction. Reaction
products were resolved on 6% urea polyacrylamide denaturing gels
followed by autoradiography.
Cells were harvested by low speed centrifugation (3,000 rpm, 30 min). Recovered supernatant was filtered through a 0.22-mm filter, and virus present in the filtrate was concentrated and purified by sucrose gradient centrifugation prior to isolation of viral RNA. HIV-1-IIIB produced from MT2/LN cells was similarly prepared for control analysis. Total viral RNA was extracted from purified viral pellets using the acid guanidinium isothiocyanate/phenol chloroform method (27). RNA concentration was determined by A260, and RNA integrity was verified by electrophoresis on a 1% agarose gel. Analysis of viral RNAs for mutant tRNAtarD was conducted by RT-PCR (see above). For each RT-PCR reaction, 0.2 µg of viral RNA was amplified by the tRNAtarD-specific primers, and amplified products were subjected to gel electrophoresis. Amplification of the viral RNAs with tRNALys-3-specific primers was performed as a control.
Amplification and Sequence Analysis of Breakthrough VirusesInfected cell DNA was extracted by the urea lysis method
(30), phenol-extracted, ethanol-precipitated, and resuspended in TE
buffer to a final concentration of 1.4 µg/µl. Target cell DNA was
diluted to contain 1 copy of proviral DNA per µl, and 1 µl of this
dilution was amplified 11 independent times with Pfu polymerase (Stratagene, La Jolla, CA) following the manufacturer's
recommendations. PCR products thus obtained were purified on ion
exchange mini-columns (QIAGEN Inc., Chatsworth, CA) and sequenced in
the sense and antisense directions (AmpliTaq, Perkin-Elmer). Primers
for amplification and sequencing were as follows: sense:
5-GGTCTCTCTGGTTAGACCAG-3
and antisense: 5
-CGTCGAGAGATCTCCTCTG-3
.
Amplification conditions were 28 cycles, where each cycle consisted of
the following steps: 94 °C for 1 min followed by 55 °C for 1 min
followed by 72 °C for 30 s.
We wished to synthesize a mutant tRNA in which
recognition of the PBS would be redirected to a region of the viral
genome different from PBS. We constructed a gene encoding a
tRNALys-3-derived mutant in which 3 primer-binding
sequences in the acceptor stem were modified to be complementary to the
highly conserved TAR stem-loop region of HIV-1. This mutant was
designated tRNAtarD (Fig. 1, panel A). This
modification was introduced with the purpose of triggering aberrant
priming of reverse transcription at a site upstream from the natural
PBS. We chose the TAR stem-loop sequence as a target for this aberrant
priming by mutant tRNA because it is a conserved sequence and is
important in the reverse transcription process itself (31). Priming of
reverse transcriptase at the TAR site would be predicted to interfere
with the retroviral life cycle by causing premature priming upstream of
the PBS.
We substituted seven nucleotides of the 3 sequence of
tRNALys-3 to produce a sequence complementary to the
conserved TAR region of HIV-1, and made six additional substitutions in
5
sequences complementary to the 3
mutations to restore the tRNA
secondary structure (Fig. 1, panel A), in the acceptor stem
such that the mutant tRNA would retain predicted secondary and tertiary
tRNA structure and ability to bind RT (32, 33). Care was taken not to
modify other regions of the tRNA which are important for polymerase III
transcription such as "A" and "B" boxes (34), for stability,
and for interaction with HIV-1 RT (5, 6, 8).
HIV-1 RT has been reported
to specifically recognize the tRNALys-3 anticodon region
(5, 35, 36). Mutant tRNAtarD retains wild-type anticodon stem-loop
sequences (Fig. 1). To determine whether substitutions within either
the 3 primer binding sequences or the structure of the acceptor stem
of tRNA would affect the affinity of RT for mutant tRNAtarD, the
previous tRNA and wild-type tRNALys-3 were transcribed
in vitro and assayed for binding by gel retardation assay in
the presence of cold competitor tRNAs (Fig. 2). As shown in Fig. 2, binding of tRNAtarD to HIV-1 RT could be effectively competed by increasing molar concentrations of tRNALys-3
(lanes 1-6). Conversely, binding of tRNALys-3
to HIV-1 RT could be effectively competed by increasing molar concentrations of tRNAtarD (lanes 9-14). The degree of
competition that tRNAtarD and tRNALys-3 exert on each other
is identical and therefore, we conclude that tRNAtarD binds to purified
HIV-1 RT with similar affinity to that of tRNALys-3 in our
assays. Additional gel-shift experiments showed that tRNAtarD bound
efficiently to HIV-1 RT, but not to MoMuLV RT (data not shown). The
ability of tRNALys-3-derived mutant tRNAtarD to bind RT is
likely retained due to the preserved anticodon region of tRNA, which is
important in recognition by HIV-1 RT (36).
Transduction of MT-2 Cells with a Retroviral Vector Encoding tRNAtarD
We transduced the MT-2 T-cell line with the retroviral
vector, N2A-tarD (Fig. 1, panel B) containing tRNAtarD
coding sequences. Assays for polymerase III-directed transcription
in vitro using Jurkat-derived T-cell extracts, and the
N2A-tarD vector coding sequences, resulted in transcription and correct
processing of tRNAtarD (data not shown). Following G418 selection of
transduced cells, DNA and RNA were extracted from bulk G418-selected
cloned cell lines, and mutant tRNA sequences were detected using
RT-PCR. A band with the size of mutant tRNAtarD (76 nucleotides) was
detected in MT2/TARD cells (Fig. 3, panel A, lanes
1, 3, 5, and 7) but was not observed in control MT2/LN
cells (Fig. 3, panel A, lane 9). No band was detected in the
MT2/TARD cells when PCR was carried out in the absence of RT
(lanes 2, 4, 6, and 8), indicating that the
target band obtained in the MT2/TARD cells was derived from tRNA, and
not residual DNA contamination. In contrast, a characteristic band
resulting from tRNALys-3 was detected with similar
intensity in both parental MT-2 and MT2/TARD cells, when the RNA
samples were amplified with tRNALys-3 primers (data not
shown). Northern blotting and hybridization with a radiolabeled
tRNAtarD-specific probe confirmed tRNAtarD expression (data not
shown).
Effects of tRNAtarD on HIV-1 Replication
To examine the relative sensitivity of the transduced MT2/TARD cells to HIV-1, an infectious stock of HIV-IIIB was simultaneously titered in both control MT2/LN cells and G418-resistant MT2/TARD cell clones (Fig. 3, panel B) and bulk-selected G418-resistant MT2/TARD cells. The relative ability of these subclones to support HIV-1 replication was determined by the TCID50 assay. Although relative titers varied among different subclones, all subclones tested showed decreased TCID50 titers compared with control MT-2 or MT2/LN cells. As shown in Fig. 3, panel B, the TCID50 titers measured for subclones 5 and 12 were 104.8 and 105.0, respectively. These two subclones were the most refractory to HIV-1 replication, showing a marked decrease (>1.5 log) in virus titers compared with control MT-2 cells (106.4) or MT2/LN (106.5). Subclones 3 and 9, representing the least resistant clones, exhibited a lower but detectable drop (<0.3 log) in virus titers compared with control cells.
Similar data were obtained with uncloned, G418-selected MT2/TARD cells.
The frequency of syncytia formation in pooled G418-resistant MT2/TARD
cells was significantly lower (Fig. 5, panel A) than that in
parental MT-2 cells (Fig. 5, panel B) when using an
identical viral inoculum. The TCID50 titer obtained at day
5 post-infection was 104.5 in MT-2 cells as compared with
103.0 in MT2/TARD cells. The relative estimated titers
increased with time and reached 106.0 in parental MT-2
cells and 104.8 in the MT2/TARD cells, respectively, at day
10 post-infection. Thus, the estimated viral titer in pooled
G418-resistant MT2/TARD cells was reproducibly 1.0-1.5 log lower than
that of control MT2/LN cells, or MT2/LN cells not expressing mutant
tRNAtarD. The estimated viral titers did not change significantly with
extended incubation of up to 15 days.
The above results, taken together with those described in Fig. 3, panel A, demonstrate that the level of tRNAtarD expression correlated with the efficiency of HIV-1 inhibition in individual cell clones. This assay was repeated several times with similar results.
Inhibition of viral replication in cells transduced with tRNAtarD could be due to changes induced in cells during the transduction, drug selection, or cloning procedures. To rule out these possibilities, we studied the rate of growth of MT-2 and MT2/TARD cells. Comparison of growth kinetics indicated that the growth rate of MT2/TARD was indistinguishable from that observed in parental MT-2 cells and MT2/LN cells (data not shown). It was also possible that lower infectivity of MT2/TARD cells was caused by decreased expression of CD4, the primary receptor for HIV-1. We performed flow cytometric analysis in all the cell clones described, and found no appreciable differences in CD4 expression with respect to MT-2 or MT2/LN cells (data not shown). Therefore, we conclude that reduction in levels of viral replication is not due to changes in growth rate or CD4 antigen expression.
To further assess the inhibitory effect of tRNAtarD on HIV-1
replication, MT2/TARD subclone 5 was tested for its ability to support
HIV-1 replication. Following HIV-1 infection, HIV-1 p24 viral antigen
was initially detected on day 9 in the control MT2/LN cultures, and
reached a peak on day 17 (Fig. 4). In contrast, p24
antigen was not detected until day 17 in MT2/TARD cells, demonstrating a delay of approximately 8 days in p24 production compared with control
cells. Visible syncytia appeared in the control cultures at day 12 post-infection, and involved most of the cells by day 17. In contrast,
very few syncytia were observed even after 27 days post-infection in
MT2/TARD cells. Representative photomicrographs of infected MT2/TARD
and MT2/LN taken at day 15 post-infection are shown in Fig.
5. Qualitatively similar results were also observed in
CEM cells transduced with N2A-tarD relative to parental CEM cells (data
not shown). These measurements demonstrated that tRNAtarD expression
resulted in decreased HIV-1 infection as assayed by either p24
production or relative titer of the same HIV-1 stock.
Analysis of tRNAtarD Incorporation into HIV-IIIB Virions
We
next determined whether mutant tRNAtarD was incorporated into HIV-1
virions. Total viral RNA from 109 TCID50 of
infectious virus was extracted from HIV-IIIB virus propagated in
MT2/TARD cells, or from virus produced in MT2/LN cells. The results of
RT-PCR amplification of viral RNAs, designed to detect the tRNAtarD
sequence, is shown in Fig. 6. When viral RNAs were
probed with tRNAtarD-specific primers, a 76-base pair tRNAtarD product
was detected in the viral RNA sample from HIV-IIIB propagated in
MT2/TARD cells (Fig. 6, lane 2), but was not seen in viral
RNA prepared from control virions (Fig. 6, lane 1). No PCR
products were observed in controls in which either viral RNA or RT was
absent (Fig. 6, lanes 3 and 4). PCR products were
detected in both control and target viral RNAs when the
tRNALys-3 specific primers were used (lanes 5 and 6). These results indicated that mutant tRNAtarD was
incorporated into HIV-IIIB virions prepared from MT2/TARD cells.
Antisense Effect of tRNAtarD on Viral Replication
A previous report demonstrated that the TAR loop was a suitable target for inhibition of HIV-1 replication using antisense RNA (37). It is possible that tRNAtarD inhibits HIV replication by binding to the TAR loop in viral mRNA, therefore interfering with Tat-mediated transactivation. A second possibility is that binding of tRNAtarD to the TAR loop on viral mRNAs would prevent their efficient expression. We tested these possibilities by measuring basal and Tat-induced transcriptional activities of the HIV-1 LTR in the presence or the absence of tRNAtarD (Table I). MT-2 cells were stably transfected with the LN (MT2/LN) or N2A-tarD (MT2/TARD) vectors. These cells were then transiently co-transfected with a construct in which the chloramphenicol acetyltransferase reporter gene was driven by the HIV-1 LTR, and with a Tat expression vector (pSV-Tat) or control plasmid. Basal LTR activity in the absence of Tat was about 2-fold higher in MT2/LN than in MT2/TARD cells, while Tat transactivation efficiencies were 23.4-fold in MT2/LN cells and 21.6-fold in MT2/TARD cells. The above experiments suggest that expression of tRNAtarD did not markedly influence in the ability of Tat to transactivate the LTR. However, we cannot exclude a moderate inhibitory effect on LTR-directed transcription. Since the expression of tRNAtarD can inhibit viral production by several orders of magnitude, we conclude that potential antisense effects exerted by tRNAtarD may account only in part for the overall virus inhibition we observed.
|
We
anticipated that tRNAtarD would interfere with normal initiation of DNA
synthesis. Consequently, we measured the ability of tRNAtarD to prime
HIV-1 RT-directed reverse transcription in vitro from a
segment of HIV-1 genomic RNA (RNA 1). The RNA 1 segment used includes
sequences from the PBS to the 5 end of the viral RNA. Reactions were
carried out in vitro with highly purified HIV-1 RT, using
in vitro synthesized tRNAtarD or tRNALys-3 as
primers. Control reactions were also carried out using three DNA
primers (Fig. 7, panel A). Expected extension
products of lengths 368, 237, and 200 from DNA primers 1, 2, and 3, respectively, were observed (Fig. 7, panel B, lanes 4, 3,
and 2). Extension of the control primers to correct lengths
demonstrates that RNA 1 can serve as an effective template for DNA
synthesis primed from a variety of locations. Extension of the tRNA
primer at the PBS would be expected to yield a product 416 nucleotides
in length, whereas priming at the TAR loop homology site would generate
a 243-nucleotide-long product. As expected, priming with
tRNALys-3 produced a 416-nucleotide-long product (Fig.
8, panel B, lane 5). Priming with tRNAtarD
also produced a 416-nucleotide band, indicating that, despite the
introduction of mutations in tRNAtarD, the residual complementarity was
sufficient to prime reverse transcription at the PBS (Fig. 7,
panel B, lane 6). A band is also visible at about position
243, consistent with extension of tRNAtarD from its intended binding
site on the TAR stem-loop. However, the observed band could also have
been caused by normal pausing of polymerization during synthesis of the
416-nucleotide product. One potential effect of the observed priming by
tRNAtarD at the PBS would be to introduce foreign sequences into the
PBS of viral progeny.
To improve our ability to see potential products of priming from the intended tRNAtarD-binding site, we measured synthesis on a shorter viral RNA template containing the TAR loop sequences but not the PBS (RNA 2). We observed a 243-nucleotide-long product consistent with priming within the stem of the TAR hairpin (Fig. 8, panel D, lane 2). No extension products were observed in control experiments containing either tRNALys-3 or DNA primer 1 plus RNA 2, since neither primer was expected to bind the template (Fig. 7, panel D, lane 1 and panel C, lane 1). Products of expected lengths were observed using DNA primers 2 and 3 on RNA 2 (Fig. 7, panel C, lanes 3 and 2). This result demonstrates that RT-directed priming can also occur from a site outside of the PBS. During infection, this would produce a short reverse transcript, likely to disrupt viral replication.
Sequence Characterization of PBS Sequences from Breakthrough Viruses Reveals the Absence of Mutations in PBS and PBS-proximal AreasWe demonstrated that exponential growth of HIV-1 in tRNAtarD-expressing cells was delayed by 7-14 days with respect to viral growth in parental MT-2 cells (Fig. 4). Although this delay is a clear indication of the inhibitory activity of the mutant tRNAtarD, significant levels of viral burden were ultimately achieved in these cultures (Fig. 4). The rise in p24 levels observed at day 17 in MT2/TARD cells, to which we will refer as "breakthrough virus," may be due to the generation of escape mutants. An alternative explanation is that viral amplification beyond a certain threshold may overwhelm the inhibitory capacity of the mutant tRNA.
We examined the possibility of mutations in the PBS and PBS-proximal areas reported to be involved in interactions with tRNALys-3 (12-18) in breakthrough viruses. Breakthrough virus was generated by infecting MT2/TARD with a HIV-1 NL4-3, a previously described molecular clone (24) at a low multiplicity of infection (0.01) and passaged until breakthrough was observed. The growth kinetics of this infection was compared with that of unmodified MT-2 cells as was described in Fig. 4. Kinetics of viral replication of HIV-1 in MT2 and MT2/TARD cells in this experiment paralleled our previous observations (Fig. 4). After 5 weeks of passage, a cell-free virus stock from the MT2/TARD cell culture was prepared. This virus stock was used to infect a fresh culture of MT2/TARD cells, this time at high multiplicity of infection (1.0) and cellular DNA was extracted 48 h later and used as a substrate for limiting-dilution PCR and DNA sequencing.
Sequence for 11 PCR amplification products analyzed comprised nucleotides 500-685 (see Fig. 8) of the HIV-1 NL4-3 proviral sequence (24). This region included the PBS and short sequences outside the PBS proposed to interact with tRNALys-3 (12-18). Sequences for 11 independent PCR clones showed no mutations when compared with the sequence of the virus used as inoculum. Therefore, we conclude that the ability of HIV-1 to replicate actively after long-term passage in MT2/TARD cells is not due to mutations in the PBS or PBS-proximal areas. It is therefore possible that mutations elsewhere in the genome confer resistance to the inhibitor tRNAtarD. However, it is also possible that virus amplification beyond the inhibitory capacity of tRNAtarD occurs after several weeks of passage, in the absence of mutations. Finally, mutations in the RT enzyme which decrease affinity for mutant tRNAtarD have not been ruled out.
tRNA functions in living cells mainly as a vehicle to translate
genetic information stored in mRNA into amino acid sequence in
proteins. Cellular tRNAs are recognized by many cellular proteins including 5 and 3
tRNA processing enzymes (38) and tRNA aminoacyl transferases (aa-tRNA synthetase) (32, 33). Most of these enzymes
recognize both the anticodon region and specific features of the
tridimensional structure of tRNA (32). There are two different human
tRNALys genes. One of these, tRNAlys-UUU (21, 39), has
complementary sequence to the HIV-1 RNA genome, and is the primer for
HIV-1 reverse transcription.
We have introduced mutations into tRNAlys-UUU (tRNALys-3)
designed to alter PBS binding specificity, while maintaining conserved sequence in the so-called A and B boxes needed for polymerase III-directed transcription (34), and maintaining integrity of the
anticodon region. We have substituted sequences in the acceptor stem,
to make a 3 domain complementary to the conserved HIV-1 TAR region. To
ensure efficient transcription of this mutated gene, we included
5
-flanking sequences which may contain enhancers for polymerase
III-directed transcription. Also included were 3
-flanking sequences
consisting of the stop signal for polymerase III, and nucleotides
required for processing, derived from the most efficiently expressed of
the three cellular tRNAlys-UUU loci (21, 40). The mutant tRNA was also
designed to maintain essential features, in regions away from the 3
end, required for interaction with HIV-1 RT and NCp7 (5, 36).
Expression of mutant tRNAtarD in cultured T-cells did not alter
cellular morphology, the rate of cell division, or CD4 antigen
expression, suggesting it did not interfere with the function of the
cellular tRNALys-3.
Mutant tRNAtarD expression results in decreased HIV-1 replication, as assayed by p24 levels, or by relative TCID50 titer in the MT-2 and CEM T-cell lines. Analysis of multiple transduced subclones showed a correlation between levels of tRNAtarD expression and HIV-1 inhibition.
Although the exact mechanism by which mutant tRNA expression protects cells against HIV-1 challenge is not yet known, several possibilities exist. Several reports indicated that p66 of the HIV-1 RT p51/p66 heterodimer recognizes and binds to the tRNALys-3 anticodon region (7, 18, 41, 42) and may help unwind the acceptor stem (18) in the presence of NCp7 protein (43). Another report demonstrated that excess wild-type tRNALys-3 primer inhibited the DNA polymerase activity of a recombinant HIV-1 RT, p66/p51 heterodimeric form (44). This effect was ascribed to the anticodon region of tRNALys-3 primer (44). As mutant tRNAtarD levels appear to be lower than endogenous tRNALys-3, direct competition is not likely to be the primary mechanism for inhibition of viral replication.
Although tRNAtarD can bind to RT, we had originally expected that it
would not prime reverse transcription from the correct PBS since
tRNAtarD lacks significant complementary sequences to the 5 region of
the HIV-1 PBS (Fig. 9). However, results of priming assays carried out in vitro using purified HIV-1 RT
demonstrate that the tRNAtarD can prime at either the PBS or the
intended binding site in the TAR stem-loop. Significantly, the three
3
-most nucleotides in tRNAtarD (5
-CCA-3
), which are
post-transcriptionally added to all tRNAs, are complementary to the
corresponding nucleotides in the PBS. Alternatively, tRNAtarD-directed
priming from the PBS would introduce a foreign sequence into the PBS
which could interfere with the second strand transfer step of viral
replication. In this step, the sequences copied into DNA from the
modified region of tRNAtarD have to bind to sequences complementary to the normal PBS sequence to complete synthesis of the double stranded DNA intermediate of viral replication. Introduction of a foreign sequence into the PBS could have additional effects, such as lowering the efficiency of priming by normal tRNALys-3 in the
altered virus. This could affect both replication in infected cells,
and the ability of the virus to infect new cells. tRNAtarD-directed priming from the TAR sequence would make a viral minus strand DNA that
is too short to complete synthesis. Aberrant cDNA synthesis could
also lead to premature viral RNA template degradation by the RNase H
activity of HIV-1 RT, in theory leading to production of
integration-defective proviral DNA.
Inhibitory effects may be enhanced further by the selective packaging of this mutant tRNA into virions, since the PBS is not thought to be required for selective incorporation of tRNALys-3 (42, 45) and since incorporation into the virions is thought to be mediated by interaction of Pr160-polymerase precursor with the tRNA anticodon region (3). We demonstrated the incorporation of mutant tRNAtarD into virions. We also observed decreased infectivity of virus particles produced in cells expressing mutant tRNAtarD compared with wild-type virions, when normalized for p24 levels (data not shown). Mutant tRNAs with homology to sequences other than the PBS may result in the production of defective virions.
Potential mutations leading to adaptation to growth in MT2/TARD cells include alterations in the PBS to acquire complementarity to TAR, mutations in TAR to reduce complementarity to mutant tRNAtarD, or mutations within RT to decrease affinity for mutant tRNA. Because results in vitro suggested that tRNAtarD was preferentially priming DNA synthesis at the PBS (Fig. 7), we wanted to ascertain whether the appearance of breakthrough viruses might be a result of mutations in the PBS. Sequence analysis of 11 independent PCR clones showed absence of mutations in the PBS and adjacent areas. This result may have several explanations. First, mutations elsewhere in the genome (i.e. the TAR region, RT coding sequence) may allow viruses to escape the inhibition by tRNAtarD. It is also possible that the production of such mutations does not occur, and the high levels of p24 production in MT2/TARD cells at late time points is a result of virus amplification beyond the inhibitory capacity of tRNAtarD. In this case, one would conclude that the inhibition of viral replication by tRNAtarD is simply more effective at low virus titers (i.e. early in infection) than at high virus titers.
We did not observe adverse effects of tRNAtarD expression on human T-cell lines as assayed by morphologic examination, CD4+ expression, or changes in growth kinetics. Although we believe the alterations we have introduced will preclude interactions with cellular aminoacyl transferase, the safety of this approach remains to be established. Rare neurologic syndromes have been described in patients with mutations in mitochondrial tRNALys-3 (46-48). Whether hematopoietic or lymphoid survival will be negatively affected in vivo by introduction of tRNAtarD is not known. However, preliminary results in T-cell lines suggest that there is no apparent toxicity, and that tRNAtarD may be useful as an anti-HIV-1 therapeutic strategy.
We have observed a major inhibition of HIV-1 replication in cells expressing mutant tRNAtarD. This strategy may offer advantages relative to conventional antisense because of the specific interaction of HIV-RT with tRNALys-3 derivative molecules, and the apparent ability of modified tRNAs to interfere with reverse transcription. The use of tRNALys-3 mutants with altered primer binding specificity to target HIV-1 replication may represent a novel gene therapy approach for acquired immunodeficiency syndrome.
We thank Dr. Eli Gilboa for the N2A vector; Dr. Arnold Berk for assistance with polymerase III transcription; Cristina Ruland, Guangqiang Wang, and Jeffery P. Morgan for technical assistance; and Dr. Alexander Black, B. Hartzog, and B. J. Rimel for review and preparation of the manuscript and figures.