Potent and Stable Attenuation of Live-HIV-1 by Gain of a
Proteolysis-resistant Inhibitor of NF-
B (I
B-
S32/36A) and
the Implications for Vaccine Development*
Ileana
Quinto
¶,
Massimo
Mallardo
,
Francesca
Baldassarre
,
Giuseppe
Scala
,
George
Englund**, and
Kuan-Teh
Jeang**
From the
Dipartimento di Biochimica e Biotecnologie
Mediche, Università degli Studi di Napoli "Federico II",
Napoli I-80131, Italy, the ** Dipartimento di Medicina
Sperimentale e Clinica, Università degli Studi di Catanzaro,
Catanzaro I-88100 Italy, and the
Laboratory of Molecular
Microbiology, NIAID, National Institutes of Health,
Bethesda, Maryland 20892-0460
 |
ABSTRACT |
Live-attenuated human immunodeficiency viruses
(HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS)
vaccine. Based on the simian immunodeficiency virus (SIV) model for
AIDS, loss-of-function (e.g. deletion of accessory genes
such as nef) has been forwarded as a primary approach for
creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably,
recent evidence suggests that loss-of-function alone is not always
sufficient to prevent the emergence of virulent mutants. New strategies
that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a
novel approach for attenuating HIV-1. We have constructed an HIV-1
genome carrying the cDNA of a proteolysis-resistant nuclear factor-
B inhibitor (I
B-
S32/36A) in the nef region.
HIV-1 expressing I
B-
S32/36A down-regulates viral expression and
is highly attenuated in both Jurkat and peripheral blood mononuclear
cells. We provide formal proof that the phenotypic and attenuating
characteristics of I
B-
S32/36A permit its stable maintenance in a
live, replicating HIV-1 despite 180 days of forced ex vivo
passaging in tissue culture. As compared with other open-reading frames
embedded into HIV/SIV genome, this degree of stability is
unprecedented. Thus, I
B-
S32/36A offers proof-of-principle that
artifactually gained functions, when used to attenuate the replication
of live HIV-1, can be stable. These findings illustrate
gain-of-function as a feasible strategy for developing safer
live-attenuated HIVs to be tested as candidates for AIDS vaccine.
 |
INTRODUCTION |
Live-attenuated human immunodeficiency virus, type-1
(HIV-1)1 viruses are vaccine
candidates for AIDS based on the assumption that poorly replicating
viruses are reduced in cytopathicity while eliciting an efficient
immune response in vivo. In fact, in the SIV model of AIDS,
viruses deleted of accessory genes, such as nef, are
attenuated and confer immune protection in adults (1-6). Recent
results have raised concerns about the safety of such live-attenuated viruses because of the emergence of virulent mutants (7-11). Hence, it
is reasonable that new mechanistically distinct strategies should be
considered in combination with loss-of-function to develop safe
live-attenuated vaccine candidates. Stable attenuation of lentivirus is
difficult. Reasons for this include the high mutability of retroviruses
as a consequence of poor fidelity in reverse transcription and high
rates of viral replication. Virus replication is one of several
parameters used to measure HIV disease progression. To this end,
endowing the viral genome with an inhibitory function that represses
replication is, in principle, a valid strategy for attenuation.
AIDS pathogenesis involves complex host-pathogen interactions (12-17).
HIV-1 entry occurs through binding of gp120 envelope glycoprotein to
CD4 and chemokine co-receptors (18, 19). Later, after reverse
transcription and integration, regulated HIV-1 gene expression is
dependent on cellular transcription factors and on the viral Tat
protein acting through several cis regulatory sequences in
the HIV-1 LTR (20, 21). Prominent among these sequences are two NF-
B
sites located upstream of the Sp1-TATAA motif (22) and a third NF-
B
site co-incident with the TAR sequence (23). The prevalence of NF-
B
sites in all HIV isolates (24) suggests that NF-
B function is
fundamentally important for virus replication and represents an
attractive target to attenuate HIVs. NF-
B is regulated by I
B
inhibitors (25). In response to activating stimuli, I
B proteins
become phosphorylated, ubiquinated, and degraded by proteasomes. This
releases active NF-
B complexes leading to transcriptional activation
of responsive genes. An I
B-
mutant protein, I
B-
S32/36A
(defective for serine 32- and serine 36-phosphorylation), was
previously shown to resist proteolysis (26, 27). I
B-
S32/36A was
found to be a particularly potent inhibitor of
NF-
B-dependent gene transcription (26, 27). Based on the
role of NF-
B in virus transcription, we reasoned that expression of
I
B-
S32/36A could be one way to attenuate HIV-1.
Here, we have investigated the feasibility of a stable bimodal
attenuation of HIV-1 through both gain- and loss-of-function. We show
that a proteolysis-resistant dominant negative I
B-
molecule (I
B-
S32/36A) can be inserted into nef of HIV-1, is
maintained in the viral genome despite prolonged passaging in tissue
culture, and contributes to a strong attenuation of HIV-1. These
results indicate that gain-of-function should be considered together
with loss-of-function in strategies for constructing safe
live-attenuated HIV-1 vaccine candidates. In addition, they demonstrate
that NF-
B inhibition is a major target for HIV-1 attenuation.
 |
EXPERIMENTAL PROCEDURES |
Construction of I
B-
Recombinant HIV-1
Viruses--
pNL
nef was generated by replacing the first 117 nucleotides of nef sequence of pNL4-3 (28) with a linker
specifying for the unique restriction sites XhoI,
XbaI, and NotI. This resulted in the frameshift
of the remaining nef sequence. cDNAs for I
B-
and
I
B-
S32/36A were amplified by PCR using a 5'-primer flanked by
SalI site and a 3'-primer flanked by the FLAG sequence
followed by stop codon and XbaI site. The 5'-primer
was 5'-ACGCGTCGACATGTTCCAGGCGGCCGAGCGC-3'; the 3'-primer was
5'-GCTCTAGATCACTTGTCGTCATCGTCTTTGTAGTCTAACGTCAGACGCTGGCCTCCAAA-3'. The PCR products for I
B-
and I
B-
S32/36A were digested
with SalI and XbaI and ligated into pNL
nef to
generate pNLI
B-wt and pNLI
B-M, respectively. I
B-
S32/36A was
also cloned in antisense orientation using a 5'-primer flanked by
XbaI site and a 3'-primer flanked by SalI site to
generate pNLI
B-as. Molecular clones were confirmed by DNA
restriction analysis and DNA sequencing.
Transfections and Viral Stocks--
293T cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% v/v
heat-inactivated fetal bovine serum and 3 mM glutamine.
Viral stocks were produced by transfecting 293T cells (5 × 106) with viral plasmids (10 µg) using calcium phosphate.
Forty hours later, the cell culture supernatant was passed through a
0.45-µm filter and measured for RT activity.
Immunoblot Analysis--
293T cells were transfected with viral
plasmids (10 µg) and were lysed in RIPA buffer 24 h later.
Proteins (10 µg) were separated by electrophoresis in 10%
SDS-polyacrylamide gel and transferred to Immobilon-P (Millipore).
Filters were blotted with AIDS patient serum or with anti-FLAG
monoclonal antibody (Eastman Kodak, Rochester, NY) and anti-I
B-
antiserum recognizing the amino acids 51-64 of I
B-
(gift from N. Rice, National Cancer Institute-Frederick Cancer Research and
Development Center, MD) using Western-Light Chemiluminescent Detection
System (Tropix, Bedford, MA).
RNase Protection Assay--
293T cells were transfected with
viral plasmids (10 µg) using calcium phosphate, and total RNA was
isolated by acid guanidinium thiocyanate/phenol/chloroform extraction 3 days after transfection. RNA was treated with RQ1 DNase from Promega (2 units/50 µg of RNA) at 37 °C for 15 min, extracted by
phenol:chloroform (25:24) and precipitated in ethanol. RNase protection
assay was performed using RPA II kit (Ambion, Austin, TX). Briefly,
aliquots of 10 µg of RNA from different samples were hybridized with
105 cpm of HIV-1 and actin RNA probes for 15 h at
44 °C. Yeast RNA was used as control. Samples were treated with
RNase A (2.5 units/ml) and RNase T1 (100 units/ml) for 30 min at
37 °C, precipitated in ethanol, resuspended in gel loading buffer
(95% formamide, 0.025% xylene cyanol, 0.025% bromphenol blue, 0.5 mM EDTA, 0.025% SDS), and separated by electrophoresis on
8 M urea, 8% acrylamide gel in Tris borate-EDTA. HIV-1
antisense probe to the pNL4-3 sequence from +350 to +535 nt
corresponding to NF-
B, Sp1, TATAA, and TAR sequences of HIV-1 LTR
was transcribed from the T7 promoter in the pGEM LTR vector. Protected
HIV-1-specific bands of 80 and 185 bp correspond to the 5'-LTR (+455 to
+535 nt of pNL4-3 sequence) and the 3'-LTR (+9425 to +9610 nt of
pNL4-3 sequence), respectively. Actin antisense probe was transcribed
using T7 RNA-polymerase from vector pTRI-
-actin-125-human (Ambion).
Protected actin transcript corresponds to a band of 127 bp. T7 RNA
polymerase-derived transcripts were produced by using the Riboprobe
in vitro Transcription System (Promega). Transfection
efficiencies were normalized to CAT activity measured from
co-transfections with pBLCAT2 (2 µg), which contains the
cat gene fused to the herpes simplex virus tk
minimal promoter, followed by CAT assay (23).
Luciferase Assay--
Jurkat cells (2 × 106)
were co-transfected with pNL-Luc-R
E
plasmid
(29) (1 µg) and pNLI
B-as or pNLI
B-M plasmids (5 µg) using
Superfect Reagent (Qiagen, Valencia, CA). Amounts of transfected DNA
were equalized with pcDNA3.1 (Stratagene, La Jolla, CA).
Transfected cells were cultured in RPMI supplemented with 10% v/v
heat-inactivated fetal bovine serum and 3 mM glutamine with
soluble CD4 (10 µg/ml) to inhibit multiple rounds of infection. Cell
extracts were prepared at 24, 48, and 72 h post-transfection and
analyzed for luciferase activity using Luciferase Assay System (Promega).
Viral Growth--
Jurkat cells were cultured in RPMI
supplemented with 10% v/v heat-inactivated fetal bovine serum and 3 mM glutamine. PBMCs were stimulated with PHA (0.5 µg/ml)
or mAb OKT3 (1 µg/ml) in RPMI 1640 with 10% v/v heat-inactivated
fetal bovine serum for 3 days and then washed and cultured in RPMI 1640 with 10% fetal bovine serum with interleukin-2 (20 units/ml). Cells
were infected with equivalent doses of viral stocks normalized by RT
activity. Cell supernatants were collected every 3 days for RT assay;
equal volumes of fresh medium were replaced into the cultures at the same time.
RT-PCR Analysis--
Viral RNA was purified from cell culture
supernatants using QIAampViral RNA kit (Qiagen). RNA extracts (1 µg)
were reverse transcribed, and PCR was amplified using Titan One Tube
RT-PCR System (Roche Molecular Biochemicals). The I
B-
S32/36A
sequence was amplified with primers flanking the cDNA insert from
+8732 to +8749 nt (5'-primer; env region) and from +9017 to
+9034 nt (3'-primer; nef region) of pNL4-3. As control, the
integrase region was amplified with 5'-primer from +4339 to +4359 nt
and 3'-primer from +4894 to +4914 nt of pNL4-3. Amplification was
carried out according to the following protocol: incubation at 50 °C
for 30 min, preheating at 95 °C for 2 min followed by 30 cycles of
95 °C for 45 s, 58 °C for 45 s, and 72 °C for 3 min.
Flow Cytometry Analysis--
Cells were stained with
phycoerythrin-conjugated anti-p24 mouse monoclonal antibody (Ortho
Diagnostic, Raritan, NJ) and Annexin-V-Fluos (Roche Molecular
Biochemicals). Flow cytometry was performed with a Coulter Epics XL
Cytometer. Ten thousands cells were analyzed under each condition.
 |
RESULTS |
HIV-1 Expressing I
B-
S32/36A Down-regulates Viral
Expression--
To determine whether the attenuation of HIV-1 could be
obtained via cis-expression of a proteolysis-resistant
inhibitor of NF-
B, FLAG-tagged I
B-
S32/36A cDNA was
positioned in sense and antisense orientations into nef of
HIV-1 pNL4-3 generating pNLI
B-M and pNLI
B-as genomes,
respectively (Fig. 1A). As a
control, an otherwise isogenic molecular clone of pNL4-3 expressing
the proteolysis-sensitive wild-type I
B-
(pNLI
B-wt) cDNA
was also constructed (Fig. 1A). These insertions also
resulted in the deletion of 39 amino acids from the N terminus of Nef
and a translational frameshift for the remaining Nef-encoding codons.
Hence, the chimeric genomes gained an I
B-
S32/36A function coupled
with a simultaneous loss of Nef function. From these molecular genomes,
different virus stocks were generated through independent transfections
of 293T cells. Expression in the transfected cells of HIV-1 proteins
and the heterologous I
B-cDNAs was verified by immunoblotting
(Fig. 1, B and C). To assess the functional
impact of I
B-
S32/36A in the context of HIV-1 gene expression,
viral transcription in 293T cells transfected individually with
pNL4-3, pNL
nef, pNLI
B-M, pNLI
B-as, or pNLI
B-wt was
analyzed by RNase protection assays. Viral RNAs were efficiently
produced from all genomes except for the I
B-
S32/36A-expressing
virus, pNLI
B-M (Fig. 2A).
This finding points to a strong inhibition of LTR-directed
transcription by I
B-
S32/36A. A normalized simultaneous
co-transfection into cells of pNLI
B-M genome with pNL4-3 genome
fused to the luciferase reporter gene still resulted in
markedly reduced viral transcription (Fig. 2B). The
inhibitory mechanism is likely to be a dominant trans-repressive effect from the I
B-
S32/36A protein
rather than a cis-destabilizing phenomenon arising as a
consequence of an insertion into nef. This is further
supported by the fact that the insertion of I
B-
cDNA either
in antisense orientation, or as wild-type sequence does not inhibit
viral expression (Fig. 2A, lanes 7 and
8). Repressed expression would have been expected if there
were a cis insertional effect.

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Fig. 1.
Recombinant HIV-1 viruses that express
I B- . Panel
A, structure of HIV-1 genomes that express I B-
wild-type (pNLI B-wt), I B- S32/36A in sense
(pNLI B-M), and antisense (pNLI B-as) orientations, respectively.
Panel B, immunoblot analysis of total extracts (10 µg
each) from 293T cells 24 h after transfection with the indicated
HIV-1 plasmids (10 µg) using a hyperimmune AIDS patient serum.
Panel C, immunoblot analysis of total extracts (10 µg)
from 293T cells 24 h after transfection with the indicated HIV-1
plasmids (10 µg) using anti-FLAG monoclonal antibody followed by an
anti-I B- antiserum. This mixture identifies both I B- -FLAG
expressed from the viral genomes and endogenous I B- .
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Fig. 2.
I B- S32/36A
represses HIV-1 LTR-directed transcription. Panel A,
293T cells were transfected with the indicated viral plasmids (10 µg). Transfection efficiencies were normalized based on the
co-transfection with 2 µg of pBLCAT2. Total RNAs were quantitated by
RNase protection assay 72 h after transfection. Viral RNAs were
measured as 3'-LTR and 5'-LTR protected fragments. Cellular
actin-mRNA was quantitated in parallel. Lane 1, RNA
probe plus yeast RNA; lane 2, RNase treatment of RNA probe
plus yeast RNA; lane 3, RNase treatment of RNA probe plus
RNA from cells transfected only with pBLCAT2. Lanes 4-8,
RNase treatment of RNA probe plus RNA from cells transfected with
pNL4-3 (lane 4), pNL nef (lane 5), pNLI B-M
(lane 6), pNLI B-as (lane 7), and pNLI B-wt
(lane 8). Lane 9, sample as in lane 6 with a 10-fold longer exposure. CAT activities, measured as percent
acetylation of [14C]chloramphenicol per 50 µg of
protein per 3 h, from lane 3-8 were: 1.5, 1.7, 1.0, 1.6, 1.2, and 1.9, respectively. Results are representative of three
independent experiments. Panel B, Jurkat cells were
transfected with pcDNA3.1, pNLI B-as, or pNLI B-M plasmids (5 µg) together with pNL-Luc-R E reporter
plasmid (1 µg). Luciferase assay was performed in cell lysates at the
indicated time. Luciferase activity is expressed as arbitrary light
units per 100 µg of protein. Results are representative of three
independent experiments.
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Attenuation of HIV-1 Expressing I
B-
S32/36A--
Next, the
replication properties of the recombinant HIV-1 genomes were measured.
We monitored for viral growths in established (Jurkat) and primary
(PBMCs) T-cells. Based on normalized amounts of input viruses,
NLI
B-M was found to be highly attenuated in Jurkat cells when
compared with the NL
nef and NLI
B-as (Fig. 3A). A reduced replication
capacity was also observed for NLI
B-M in human PBMCs stimulated
either with anti-CD3 mAb OKT3 (Fig. 3B) or PHA (Fig.
3C). In parallel, the chimeric HIV-1 which expresses the
proteolysis-sensitive wild-type I
B-
produced a replication profile that was only slightly attenuated when compared with control NLI
B-as virus (Fig. 4,
A-C). Taken together, these results indicate a critical
contribution of proteolysis-resistant mutant I
B-
toward
attenuated viral growth.

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Fig. 3.
HIV-1 expressing
I B- S32/36A is highly
attenuated in Jurkat cells and human PBMCs. Panel A,
Jurkat (5 × 104) cells were infected with NL4-3,
NL nef, NLI B-M, and NLI B-as with equal amounts of viruses
normalized based on RT counts of 104 cpm (left)
or 103 cpm (right). Panel B, PBMCs
(2 × 105 cells) stimulated with mAb OKT3 were
infected with the indicated viruses normalized for 105 cpm
(left) or 104 cpm (right) of RT.
Panel C, PBMCs (2 × 105 cells) stimulated
with PHA were infected with viruses normalized for 104 cpm
(left) or 103 cpm (right) of RT.
Viral growth was measured by RT activity in cell culture supernatants.
Results representative of six independent infections are shown.
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Fig. 4.
Wild-type proteolysis-sensitive
I B- poorly attenuates
HIV-1 replication. Jurkat cells (panel A), PBMCs
stimulated with mAb OKT3 (panel B), and PBMCs stimulated
with PHA (panel C) were infected with NLI B-M, NLI B-as,
or NLI B-wt viruses and measured for viral production as detailed in
Fig. 3.
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Attenuation of NLI
B-M Is Not Because of
Apoptosis--
Previously, I
B-
S32/36A was shown to enhance the
apoptosis induced by tumor necrosis factor-
(30-32). Because tumor
necrosis factor-
is produced during HIV-1 infection (33, 34), we
asked whether a pro-apoptotic effect of I
B-
S32/36A might account, in part, for attenuation of viral growth. To this end, Jurkat cells
infected separately with NL4-3, NLI
B-M, or NLI
B-as viruses were
analyzed by flow cytometry for the expression of p24 and for binding to
annexin V, a marker of an early event in apoptosis (35). Because of the
different viral growth kinetics, to account fully for equivalent levels
of virus production, cell samples were analyzed at variant times
post-infection. As shown in Table I, the
percentage of apoptotic cells among p24-positive population was 17 and
13% for NL4-3 and NLI
B-as, respectively, at day 10 post-infection,
as compared with 19% for NLI
B-M at day 18 post-infection. Thus, the
expression of I
B
S32/36A did not affect apoptosis significantly because the percentage of apoptotic cells among those infected with
NL4-3, NLI
B-as, or NLI
B-M was similar.
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Table I
Flow cytometric analysis of Jurkat cells infected with NL4-3,
NLI B-as, or NLI B-M
The cells were analysed by flow cytometry for the expression of p24, as
marker of viral infection, and for binding to annexin V, as marker of
apoptosis. Ten thousand cells were analysed under each condition.
Values are expressed as percentages.
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NLI
B-M Is Stably Attenuated through Cell
Passaging--
Retroviruses rapidly delete heterologous genes which do
not confer benefits for replication (36-38). One indication of such a
loss in gained function would be a change in the kinetics of replication in later (compared with earlier) passages of viruses. In
examining NLI
B-M virus after prolonged culturing in cells, we noted
that its attenuated phenotype bred true despite extended re-passaging
(Fig. 5, A-C). No changes in
replication phenotype were observed with up to 180 days (9 passages) of
continuously forced propagation in ex vivo tissue cultures.
This stability of I
B-
S32/36A contrasts sharply with documented
findings in other systems where rapid deletion of gained function
commenced as early as 48 h after viral passaging (36). To verify
biochemically that this phenotypic stability was from the maintenance
of I
B-
S32/36A in nef, RT-PCR analysis of virion RNAs
was performed using samples isolated after various times of culturing
in Jurkat cells (Fig. 5D). Viral RNAs from first passage (20 days in culture) as compared with sixth (120 days in culture) and ninth
(180 days in culture) passage were indistinguishable, providing no
evidence for deletion of I
B-
S32/36A (Fig. 5D, compare
lanes 5-7). Thus, by contrast with other cDNAs inserted
into nef (36-38) in the setting of replicating HIV-1/SIV,
I
B-
S32/36A is unprecedently stable.

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Fig. 5.
NLI B-M virus
maintains an attenuated phenotype through serial passaging. Jurkat
(panel A), PBMCs stimulated with mAb OKT3 (panel
B), and PBMCs stimulated with PHA (panel C) were
infected with NLI B-as, NLI B-M, or passaged NLI B-M viruses
and measured for viral production as detailed in Fig. 3. NLI B-M
virus was obtained through serial passages in Jurkat cells by
collecting the RT peak of viral production at each passage. On average,
each passage was for 20 days. Panel D, RT-PCR analysis of
nef and integrase regions of NLI B-M virion
RNAs. The wild-type nef product (302 bp; lane 2),
the nef product (200 bp; lane 3), the I B- S32/36A
product (1180 bp; lanes 4-7), and the integrase product
(575 bp; lanes 9-14) were visualized by RT-PCR of viral
RNA. Lane 1, 100-bp DNA ladder (Life Technologies, Inc.);
lanes 2 and 9, amplification of NL4-3 virion
RNAs from 293T transfected with pNL4-3. Lanes 3 and
10, amplification of NL nef virion RNAs from 293T
transfected with pNL nef; lanes 4 and 11,
amplification of NLI B-M virion RNAs from 293T transfected with
pNLI B-M; lanes 5 and 12, amplification of
NLI B-M virion RNAs from first passage in Jurkat; lanes 6 and 13, amplification of NLI B-M virion RNAs from sixth
passage in Jurkat; lanes 7 and 14, amplification
of NLI B-M virion RNAs from ninth passage in Jurkat; lanes
8 and 15, RT-negative controls.
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 |
DISCUSSION |
Various approaches have been proposed for generating
live-attenuated HIV-1 (39). Based on findings from the SIV model of AIDS, loss-of-function (e.g. deletion of accessory genes
such as nef, vpr, and U3) has been forwarded as a
primary approach for creating enfeebled, but replication-competent,
HIV/SIV (1-6). Regrettably, recent evidence indicates that
loss-of-function alone is not always sufficient (and is perhaps largely
insufficient) to prevent virulence (7-11). Hence, one is faced with
either abandoning live-attenuated virus as a potential route for HIV-1
vaccine or devising another approach which is mechanistically distinct
from and could be used additively with loss-of-function in attenuating the AIDS virus. Attenuation of HIV-1 by gain-of-function was first broached four years ago (40). However, an issue that has impeded the
useful application of gain-of-function is uncertainty over the
stability of functions added to a replicating HIV-1 genome. To date,
all exogenous open-reading frames, such as thymidine kinase,
-interferon, interleukin-2 (36-38), when artifactually embedded
into the HIV-1/SIV genome have been unstable to prolonged ex
vivo passaging, suggesting a rule of rapid deletion of gained genes upon virus replication. However, because the sampling size for
gained functions has been small, it remains unclear whether this is
indeed a universal rule. Our findings from I
B-
S32/36A formally
challenge the rigidity of this rule.
An ability by viruses in general and by HIV-1 specifically to maintain
stably gained functions should not be inherently surprising. For
example, piracy of large numbers of cellular genes by herpesviruses has
been well documented (see Ref. 41 and references cited therein). In
simple animal retroviruses, only Gag, Pol, and Env open-reading frames
are required for virus propagation. Hence, the evolution of tat,
rev, nef, vpu, vif, and vpr in the existing HIV-1
genome represents likely illustrations of how this virus has anciently through natural means acquired and continues to maintain stably added
reading frames. Conceivably, I
B-
S32/36A has certain cellular characteristics that benefit the virus-cell interaction during HIV-1
replication. What might be the propitious characteristics specified by
I
B-
S32/36A await further investigation. It is possible that the
inhibition of virus transcription by I
B-
S32/36A allows the cell
to survive HIV-1 infection, while cell killing occurs in the case of
recombinant viruses that carry exogenous genes which do not confer
virus attenuation. A second possible explanation for the insert
stability in NLI
B-M is that I
B-
S32/36A could provide an
"anti-mutator" effect to the chimeric HIV-1 genome. This
"anti-mutator" effect would confer a relative resistance to the
deletion of the gained function. In this regard, the expression of a
dominant negative I
B-
has been shown to correct aberrant DNA
synthesis which causes genetic instability in ataxia telangectasia cells (42).
In conclusion, we demonstrate here the feasibility of a
gain-of-function strategy for stably attenuating live HIV-1.
Specifically, we show that the addition of I
B-
S32/36A to an HIV-1
genome conferred a highly attenuated replication phenotype that is
genetically stable for a minimum of 180 days of growth in tissue
culture. Attenuated replication in ex vivo tissue culture is
by no means a perfect predictor of in vivo viral robustness;
however, the unprecedented observation that emerges from
I
B-
S32/36A is its durability in settings where other
artifactually gained open-reading frames (36-38) have failed. An
important extension to the experiments here would be to explore the
in vivo stability of I
B-
S32/36A in chimeric SIVs
propagated in macaques. However, the fact that transcription from the
SIV LTR is not ruled by NF-
B in the same manner as that from the
HIV-1 LTR (43-45) undermines the feasibility of such a heterologous
in vivo test for I
B-
S32/36A. In this regard, further
studies on SIV LTR-driven transcription are warranted to identify
analogous cellular inhibitors of SIV replication. That not
withstanding, the findings from I
B-
S32/36A represent a first-step
proof that gain-of-function could be a stably useful approach for
attenuation. This first step should spur further stepwise exploration
for other useful attenuating markers that might be more amenable to
incremental testing in nonhuman primate models. If so, a combination
approach that incorporates gain-of-function with loss-of-function could
possibly contribute toward the development of a live-attenuated HIV-1 vaccine.
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FOOTNOTES |
*
This work was supported by Ministero della
Sanità-Istituto Superiore di Sanità-Programma Nazionale di
Ricerca sull`AIDS Grant 40A.0.85, by Pediatric AIDS Foundation Grant
PS-22055, and by the AIDS Antiviral Targeted program from the Office of
the Director, National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a fellowship from Istituto Superiore di
Sanità-Progetto di Ricerche sull'AIDS.
¶
To whom correspondence should be addressed: Dipartimento di
Biochimica E Biotecnologie Mediche, via Sergio Pansini 5, I-80131 Napoli, Italy. Tel.: 39-081-7463157; Fax: 39-081-7463150; E-mail: quinto{at}dbbm.unina.it
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus, type-1;
SIV, simian immunodeficiency virus;
AIDS, acquired immunodeficiency syndrome;
LTR, long terminal repeat;
NF-
B, nuclear factor kappa B;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
PBMC, peripheral blood mononuclear cell;
CAT, chloramphenicol acetyltransferase;
PHA, phytohemagglutinin;
nt, nucleotide(s);
bp, base pair(s);
mAb, monoclonal antibody.
 |
REFERENCES |
-
Daniel, M. D.,
Kirchhoff, F.,
Czajak, S. C.,
Sehgal, P. K.,
and Desrosiers, R. C.
(1992)
Science
258,
1938-1941[Medline]
[Order article via Infotrieve]
-
Almond, N.,
Kent, K.,
Cranage, M.,
Rud, E.,
Clarke, B.,
and Stott, E. J.
(1995)
Lancet
345,
1342-1344[CrossRef][Medline]
[Order article via Infotrieve]
-
Wyand, M. S.,
Manson, K. H.,
Garcia-Moll, M.,
Montefiori, D.,
and Desrosiers, R. C.
(1996)
J. Virol.
70,
3724-3733[Abstract]
-
Beer, B.,
Baier, M.,
zur Megede, J.,
Norley, S.,
and Kurth, R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4062-4067[Abstract/Free Full Text]
-
Shibata, R.,
Siemon, C.,
Czajak, S. C.,
Desrosiers, R. C.,
and Martin, M. A.
(1997)
J. Virol.
71,
8141-8148[Abstract]
-
Desrosiers, R. C.,
Lifson, J. D.,
Gibbs, J. S.,
Czajak, S. C.,
Howe, A. Y.,
Arthur, L. O.,
and Johnson, R. P.
(1998)
J. Virol.
72,
1431-1437[Abstract/Free Full Text]
-
Baba, T. W.,
Jeong, Y. S.,
Pennick, D.,
Bronson, R.,
Greene, M. F.,
and Ruprecht, R. M.
(1995)
Science
267,
1820-1825[Medline]
[Order article via Infotrieve]
-
Wyand, M. S.,
Manson, K. H.,
Lackner, A. A.,
and Desrosiers, R. C.
(1997)
Nat. Med.
3,
32-36[Medline]
[Order article via Infotrieve]
-
Cohen, J.
(1997)
Science
278,
24-25[Free Full Text]
-
Berkhout, B.,
Verhoef, K.,
van Wamel, J. L. B.,
and Back, N. K. T.
(1999)
J. Virol.
73,
1138-1145[Abstract/Free Full Text]
-
Baba, T. W.,
Liska, V.,
Khimani, A. H.,
Ray, N. B.,
Dailey, P. J.,
Penninck, D.,
Bronson, R.,
Greene, M. F.,
McClure, H. M.,
Martin, L. N.,
and Ruprecht, R. M.
(1999)
Nat. Med.
5,
194-203[CrossRef][Medline]
[Order article via Infotrieve]
-
Coffin, J. M.
(1995)
Science
267,
483-489[Medline]
[Order article via Infotrieve]
-
Perelson, A. S.,
Neumann, A. U.,
Markowitz, M.,
Leonard, J. M.,
and Ho, D. D.
(1996)
Science
271,
1582-1586[Abstract]
-
Fauci, A. S.
(1996)
Nature
384,
529-534[CrossRef][Medline]
[Order article via Infotrieve]
-
Finzi, D.,
and Siliciano, R. F.
(1998)
Cell
93,
665-671[Medline]
[Order article via Infotrieve]
-
Douek, D. C.,
McFarland, R. D.,
Keiser, P. H.,
Gage, E. A.,
Massey, J. M.,
Haynes, B. F.,
Polis, M. A.,
Haase, A. T.,
Feinberg, M. B.,
Sullivan, J. L.,
Jamieson, B. D.,
Zack, J. A.,
Picker, L. J.,
and Koup, R. A.
(1998)
Nature
396,
690-695[CrossRef][Medline]
[Order article via Infotrieve]
-
Marx, P. A.,
and Chen, Z.
(1998)
Semin. Immunol.
10,
215-223[CrossRef][Medline]
[Order article via Infotrieve]
-
Littman, D. R.
(1998)
Cell
93,
677-680[Medline]
[Order article via Infotrieve]
-
Rizzuto, C. D.,
Wyatt, R.,
Hernandez-Ramos, N.,
Sun, Y.,
Kwong, P. D.,
Hendrickson, W. A.,
and Sodroski, J.
(1998)
Science
280,
1949-1953[Abstract/Free Full Text]
-
Roulston, A.,
Lin, R.,
Beauparlant, P.,
Wainberg, M. A.,
and Hiscott, J.
(1995)
Microbiol. Rev.
59,
481-505[Abstract]
-
Berkhout, B.,
Silverman, R.,
and Jeang, K-T.
(1989)
Cell
53,
273-282
-
Nabel, G.,
and Baltimore, D.
(1987)
Nature
326,
711-713[CrossRef][Medline]
[Order article via Infotrieve]
-
Mallardo, M.,
Dragonetti, E.,
Baldassarre, F.,
Ambrosino, C.,
Scala, G.,
and Quinto, I.
(1996)
J. Biol. Chem.
271,
20820-20827[Abstract/Free Full Text]
-
Myers, G., Korber, B., Foley, B., Jeang, K. T., Mellors, J. W., and Wain-Hobson, S.
(eds)
(1996)
Human Retroviruses and AIDS, p. I-3, Los Alamos National Laboratory, Theoretical Biology and Biophysics Group T-10, Los Alamos, NM
-
Ghosh, S.,
May, M. J.,
and Kopp, E. B.
(1998)
Annu. Rev. Immunol.
16,
225-260[CrossRef][Medline]
[Order article via Infotrieve]
-
Brown, K.,
Gerstberger, S.,
Carlson, L.,
Franzoso, G.,
and Siebenlist, U.
(1995)
Science
267,
1485-1488[Medline]
[Order article via Infotrieve]
-
Brockman, J. A.,
Scherer, D. C.,
McKinsey, T. A.,
Hall, S. M.,
Qi, X.,
Lee, W. Y.,
and Ballard, D. W.
(1995)
Mol. Cell. Biol.
15,
2809-2818[Abstract]
-
Adachi, A.,
Gendelman, H. E.,
Koenig, S.,
Folks, T.,
Willey, R.,
Rabson, A.,
and Martin, M. A.
(1986)
J. Virol.
59,
284-291[Medline]
[Order article via Infotrieve]
-
Connor, R. I.,
Chen, B. K.,
Choe, S.,
and Landau, N. R.
(1995)
Virology
206,
935-944[CrossRef][Medline]
[Order article via Infotrieve]
-
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784[Abstract/Free Full Text]
-
Wang, C. Y.,
Mayo, M. W.,
and Baldwin, A. S., Jr.
(1996)
Science
274,
784-787[Abstract/Free Full Text]
-
Van Antwerp, D. J.,
Martin, S. J.,
Kafri, T.,
Green, D. R.,
and Verma, I. M.
(1996)
Science
274,
787-789[Abstract/Free Full Text]
-
Wright, S. C.,
Jewett, A.,
Mitsuyasu, R.,
and Bonavida, B.
(1988)
J. Immunol.
141,
99-104[Abstract/Free Full Text]
-
Poli, G.,
Kinter, A.,
Justement, J. S.,
Kehrl, J. H.,
Bressler, P.,
Stanley, S.,
and Fauci, A. S.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
782-785[Abstract]
-
Martin, S. J.,
Reutelingsperger, C. P.,
McGahon, A. J.,
Rader, J. A.,
van Schie, R. C.,
LaFace, D. M.,
and Green, D. R.
(1995)
J. Exp. Med.
182,
1545-1556[Abstract]
-
Smith, S. M.,
Markham, R. B.,
and Jeang, K. T.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7955-7960[Abstract/Free Full Text]
-
Giavedoni, L. D.,
and Yilma, T.
(1996)
J. Virol.
70,
2247-2251[Abstract]
-
Gundlach, B. R.,
Linhart, H.,
Dittmer, U.,
Sopper, S.,
Reiprich, S.,
Fuchs, D.,
Fleckenstein, B.,
Hunsmann, G.,
Stahl-Hennig, C.,
and Uberla, K.
(1997)
J. Virol.
71,
2225-2232[Abstract]
-
Letvin, N. L.
(1998)
Science
280,
1875-1880[Abstract/Free Full Text]
-
Kestler, H. W.,
and Jeang, K. T.
(1995)
Science
270,
1219[Medline]
[Order article via Infotrieve]
-
Nicholas, J.,
Ruvolo, V.,
Zong, J.,
Ciufo, D.,
Guo, H. G.,
Reitz, M. S.,
and Hayward, G. S.
(1997)
J. Virol.
71,
1963-1974[Abstract]
-
Jung, M.,
Zhang, Y.,
Lee, S.,
and Dritschilo, A.
(1995)
Science
268,
1619-1621[Medline]
[Order article via Infotrieve]
-
Ilyinskii, P. O.,
Simon, M. A.,
Czajak, S. C.,
Lackner, A. A.,
and Desrosiers, R. C.
(1997)
J. Virol.
71,
1880-1887[Abstract]
-
Zhang, J.,
Novembre, F.,
and Rabson, A. B.
(1997)
Virus Res.
49,
205-213[CrossRef][Medline]
[Order article via Infotrieve]
-
Pohlmann, S.,
Floss, S.,
Ilyinskii, P. O.,
Stamminger, T.,
and Kirchhoff, F.
(1998)
J. Virol.
72,
5589-5598[Abstract/Free Full Text]
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