Background
Whole-genome expression profiling exemplified by the
development of DNA microarrays represents a major advance
in genome-wide functional analysis [ 1 2 ] . In a single
assay, the transcriptional response of each gene to a
change in cellular state can be measured, whether it is a
viral infection, host cell cycle changes, chemical
treatment, or genetic perturbation. Specifically,
systematic approaches for identifying the biological
functions of cellular genes altered during these changes,
such as HIV-1 infection, are needed to ensure rapid
progress in defining significant host and viral genome
sequences in directed experimentation and applications.
Therefore, host cellular states can be inferred from the
expression profiles, and the notion that the global
transcriptional response constitutes a detailed molecular
phenotype, such as class discovery, class prediction, drug
target validation, and the classification of tumors by
expression profiling has begun to receive considerable
attention [ 3 4 5 6 7 8 9 10 11 ] .
Since its discovery, much of the mainstream human
immunodeficiency virus type 1 (HIV-1) Tat research has
focused on its ability to activate the HIV-1 LTR. However,
to date, besides the transactivation activity on the HIV-1
promoter, few other effects exerted by HIV-1 Tat on
cellular and viral genes has also been observed. The Tat
protein has been shown to transcriptionally repress host
cellular genes and be involved in the immunosuppression
associated with viral infection. For instance, HIV-1
infection is able to down-regulate major histocompatibility
complex type I (MHC-I) by various different viral proteins,
including Tat which represses the transcription of MHC-I,
Vpu which retains nascent MHC-I chains in the endoplasmic
reticulum, and Nef which can mediate selective
internalization of MHC-I molecules from the plasma
membrane. MHC class I gene expression has also been shown
to be reduced upon infection with the wild-type LAI virus
or a Tat exon one recombinant virus [ 12 13 ] .
Tat has been shown to down-regulate mannose receptor,
EDF-1, CD3-gamma, and TCR/CD3 surface receptor [ 14 ] . Tat
reduces mannose receptor levels and promoter activity in
mature macrophages and dendritic cells by interfering with
the host transcriptional machinery; resulting in decreased
levels of surface mannose receptor needed for Ag
(mannosylated albumin uptake) or pathogen capture
(Pneumocystis carinii phagocytosis), and eventual delivery
to MHC class II-containing intracellular compartments [ 15
] . EDF-1, a gene down-regulated when endothelial cells are
induced to differentiate
in vitro , was shown to be
down-regulated by Tat at the transcriptional level,
resulting in the inhibition of endothelial cell growth and
in the transition from a nonpolar cobblestone phenotype to
a polar fibroblast-like phenotype [ 16 ] .
When examining the
in vivo effects of HIV-1 Tat protein
in the
Xenopus embryo, it was found that
upon injection of synthetic Tat mRNA into zygotes, a marked
delay in gastrulation occurred. This led to the altered
specification of the anterior-posterior axis and partial
loss of the anterior embryo structures. Mechanistically,
HIV-1 Tat elicited a general suppression of gene
expression, including that of
Xbra and
gsc , two early genes whose
expression are required for proper gastrulation [ 17 ]
.
In relation to the cell cycle, Tat has also been shown
to bind to p53 and inhibit the transcription of p53
responsive elements, such as the p21/Waf1 gene promoter.
Consequently, upon introduction of stress signals (e.g.,
gamma irradiation), HIV-1-infected cells lose their G1/S
checkpoint, enter the S-phase inappropriately, and apoptose
[ 18 19 20 ] . Finally, the inhibition of Tat on
translational machinery has also been noted. The potential
translational inhibitory effects of the TAR RNA region is
mediated by the activation of p68 (the interferon-induced
68-kilodalton protein kinase) kinase, which was
down-regulated by Tat during productive HIV-1 infection [
22 ] .
Although the mechanism of the host cellular
down-regulation remains largely unknown, few reports have
attempted to decipher the mechanism of the observed
inhibition. For instance, the addition of Tat to PC12 cells
up-regulated the expression of the inducible cAMP early
repressor (ICER), a specific member of the cAMP-responsive
element modulator transcription factor family, in a
cAMP-dependent manner. In turn, ICER overexpression
abrogated the transcriptional activity of the TH promoter,
strongly suggesting ICER's involvement in Tat-mediated
inhibition of gene expression [ 23 ] .
Aside from induction of ICER, Tat is capable of forming
complex (es) with a component of TFIID, TAF
II 250 [ 24 ] and Tip60 [ 25 ] both of
which contain histone acetyltransferase (HAT) activity. In
these cases, Tat-TAF
II 250 and Tat-Tip60 do not affect the
transcription from the HIV-1 LTR, but interfere with the
transcription activity of cellular genes. It is postulated
that different targets of HATs by Tat have different
consequences. The interaction of Tat with p300/CBP and
P/CAF stimulates its ability to transactivate LTR-dependent
transcription, while Tat-TAF
II 250 or Tat-Tip60 interactions control
the transcription of cellular genes.
Here to better understand the host response to Tat, we
have performed microarray experiments on HIV-1 infected
cells expressing the Tat protein. To our surprise many host
cellular genes were down-regulated when comparing HIV-1
infected latent cells to uninfected parental cells. Because
most, if not all, latent infected cells available to date
(e.g., ACH2, U1, J1.1, OM.10) have various expression
levels of doubly spliced viral mRNAs, including Tat, Rev,
Nef, Vpr, and other accessory proteins, we decided to
perform the microarray in a system where Tat was
constitutively expressed; asking whether Tat by itself, or
in the absence of other accessory proteins, could still
down-regulate host cellular genes. Consistent with latently
infected cells, we found many cellular genes to be
down-regulated in Tat expressing lymphocytes. The
down-regulation is most apparent on cellular receptors that
have intrinsic receptor tyrosine kinase (RTK) activity and
signal transduction members that mediate RTK function;
including the Ras-Raf-MEK pathway, and co-activators such
as p300/CBP and SRC-1, which mediate gene expression
related to hormone receptor genes. Interestingly, we also
observed up-regulation of S-phase genes, as well as
ribosomal genes involved in translation. Functionally,
down-regulation of receptors may allow latent HIV-1
infected cells to either hide from the immune system or
avoid extracellular differentiation signals normally
regulated by receptors. Up-regulation of S-phase and
translation genes may allow speeding of cells through the
S-phase and subsequent accumulation at the G2 phase, where
most of the cellular and viral translation may take place.
Therefore, the presence of Tat may not only control
activated transcription on HIV-1 LTR, but also aid in the
subsequent translation of viral mRNA in the cytoplasm.
Results and discussion
Receptor family members
It has long been known that infection by HIV-1
commonly leads to the down-regulation and the
disappearance of CD4 receptors from the plasma membrane,
a phenomenon referred to as receptor down-modulation.
This, in turn, renders cells refractory to subsequent
infection by the same or other viruses that use the CD4
receptor for entry; thus creating a state of
super-infection immunity. Results in Table 1indicate that
although few receptor genes were up-regulated, most of
the cellular receptors in general, were down-regulated in
the presence of the Tat protein. Most of these receptors
or membranous proteins were initially discovered from
immune or neuronal cells, hence they were given names
related to the immune or nervous system. For instance,
mRNA for the neuropeptide Y-like receptor (Acc# X71635),
which was up-regulated in Tat expressing cells, was
initially discovered as a G-protein coupled neuropeptide
Y receptor, and later found to be homologous to the
co-receptor CCR5 needed for HIV-1 infection of
monocyte/macrophage cells. Therefore, most of the
receptors listed in Table 1may in fact be expressed in
various tissues and have multiple functions.
Consistent with our microarray results on CCR5
up-regulation, experiments performed in peripheral blood
mononuclear cells (PBMCs) with soluble Tat has shown
selective entry and replication of CCR5 virus into cells
[ 27 28 ] . Up-regulation of HIV-1 coreceptor by Tat has
also been reported, where a synthetic Tat protein that
was immobilized on a solid substrate, up-regulated the
surface expression of the chemokine receptors in purified
populations of primary resting CD4+ T cells. Also, a
similar result was seen from Tat protein actively
released by HIV-1 infected cells, implying a potentially
important role for extracellular Tat in rendering the
bystander CD4+ T cells more susceptible to infection [ 28
] .
We therefore tested whether H9/Tat cells, which showed
an increase in CCR5 expression, could in fact allow
better entry and infection of the CCR5 (R5) virus into
cells. Figure 3Ashows the result of such an experiment,
where H9/Tat cells allowed a better replication profile
of the R5 than the CXCR4 (X4) virus. The increase in
viral titer peaked after some 18 days of infection with
the R5 virus, further implying that the CCR5 co-receptor
allowed a better selection of R5 virus in Tat expressing
cells.
Another example of co-receptors with multiple
functions is the leukotriene family member B4, which was
down-regulated in Tat expressing cells (Acc# D89078,
Table 1). The cysteinyl leukotrienes (CysLT), LTC, LTD,
and LTE, were first shown to be essential mediators in
asthma [ 29 ] . However, when the mouse leukotriene B4
receptor (m-BLTR) gene, was cloned it was shown to have
significant sequence homology with chemokine receptors
(CCR5 and CXCR4), co-receptors for many different HIV-1
clades [ 30 ] . Along the same lines, when cells were
infected with 10 primary clinical isolates of HIV-1,
leukotriene B4 receptor was primarily utilized for
efficient entry into cells which were mainly of the
syncytium-inducing phenotype [ 31 ] . Therefore,
up-regulation of neuropeptide Y-like receptor and
down-regulation of leukotriene B4 receptor in Tat
expressing cells indicates a selective advantage of one
class of virus (CCR5) over another (CXCR4).
Other examples of consistency between our microarray
results on receptors and the HIV-1 Tat literature,
include the down-regulation of gene expression in uPAR
(Acc# X74039), IP3 (Acc# D26070, D26351), Glu R flop
(Acc# U10302), PPAR (Acc# L07592), alpha-2 macroglobulin
receptor protein (Acc# M63959), and receptor tyrosine
kinase (Acc# L36645, U66406) genes.
The transmembranous urokinase-type plasminogen
activator receptor (uPAR; CD87) focuses the formation of
active plasmin at the cell surface, thus enhancing
directional extracellular proteolysis. Interestingly, the
promoter activity of the CD87 gene was shown to decline
after infection [ 32 ] , implying that post integration
of HIV-1 may in fact down-regulate CD87 gene expression.
Similarly, inositol 1,4,5-trisphosphate receptors (IP3R)
are intracellular calcium release channels involved in
diverse signaling pathways and are required for the
activation of T lymphocytes [ 33 ] . Tat (also implicated
as a neurotoxin) has been shown to release calcium from
inositol 1,4, 5-trisphosphate (IP3) receptor-regulated
stores in neurons and astrocytes causing premature
apoptosis [ 34 ] . Down-regulation of IP3 may therefore
contribute to viral latency and maintenance of an
anti-apoptotic state in cells.
HIV-1 infection can cause extensive neuronal loss and
clinically, a severe dementia. The cause of the
neurotoxicity remains unclear as neurons are not
infected, but the disturbance of glutamate-linked calcium
entry has been implicated. It has been shown that
HIV-infected brain has a decrease of mRNA and protein of
the GluR-A flop subtype of
alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) glutamate receptor in cerebellar Purkinje cells.
The observed disturbance of AMPA receptors may contribute
to the neurotoxic process in other vulnerable brain
regions and clinically to the development of dementia [
35 ] . Interestingly, in a mouse model AMPA receptors in
the cortex, striatum, hippocampus, and cerebellum
declined by 29-50% as early as 8 weeks post-retroviral
inoculation. Thus, the reduction in AMPA receptor density
may contribute to the development of the cognitive
abnormalities associated with HIV-1 infection [ 36 ]
.
Finally, patients with AIDS who are receiving therapy
with HIV-1 protease inhibitors have been reported to be
afflicted with a syndrome characterized by lipodystrophy
(fat redistribution favoring the accumulation of
abdominal and cervical adipose tissue), hyperlipidemia,
and insulin resistance. Potential mechanisms for altered
adipocyte function include, direct binding to PPARgamma
or inhibition of transcription of PPARgamma promoter [ 37
] . The lipodystrophy syndrome may be a result of the
inhibition of 2 proteins involved in lipid metabolism
that have significant homology to the catalytic site of
HIV proteases; namely cytoplasmic retinoic acid binding
protein type 1 and low density
lipoprotein-receptor-related protein [ 38 ] . An
additional mechanism of PPAR down-regulation may be
related to Tat expression in latent cells.
Translation associated factors
Viruses have evolved a remarkable variety of
strategies to modulate the host cell translation
apparatus with the aim of optimizing viral mRNA
translation and replication. For instance, viruses
including Herpes simplex virus type 1 (HSV-1) have been
known to induce severe alterations of the host
translational apparatus, including the up-regulation of
ribosomal proteins and the progressive association of
several nonribosomal proteins, such as VP19C, VP26, and
the poly(A)-binding protein 1 (PAB1P) to ribosomes [ 39 ]
. In the case of HIV-1, approximately one infectious
HIV-1 genome in an infected cell could be transcribed and
translated into 50,000 to 100,000 physical particles [ 40
] . This poses an immense challenge for the virus to be
able to transcribe, splice, transport, and translate its
RNA into fully packaged virions in a timely fashion.
Therefore, it would be advantageous for the virus to set
the stage for each successive step necessary for viral
progeny formation. One such event is Tat's ability to
control genes that aid in translational machinery. As
seen in Table 2, many of the critical components of a
functional ribosome, including large subunits L 3, 6, 26,
31, 38, and 41, as well as S 6, 12, 20, and 24, and many
of the translation initiation factors are up-regulated by
Tat. This would imply that Tat up-regulates many
ribosomal genes that may be necessary to produce
functional ribosomes needed for viral mRNA translation.
Therefore, interfering with translation could provide a
new strategy for anti-HIV treatment. Along these lines,
when the aminogylcosides (kanamycin, hygromycin B,
paromycin and neomycin) due to their ability to inhibit
protein synthesis by affecting ribosomal fidelity, or
puromycin because of its competing ability with tRNAs for
binding on the large ribosomal subunit, or cycloheximide
which inhibit the large ribosomal subunit by preventing
ribosomal movement along the mRNA, were used in active
HIV-1 infection, it was found that both cycloheximide and
puromycin produced the greatest decrease in HIV-1
inhibition, presumably by inhibiting the large subunit of
the ribosome [ 41 ] .
Translation of HIV-1 RNAs pose a challenge since they
all contain a TAR sequence at their 5' end. The
Tat-responsive region (TAR) of HIV-1 exhibits a
trans-inhibitory effect on translation by activating the
interferon-induced 68-kilodalton protein kinase.
Productive infection by HIV-1 has been shown to result in
a significant decrease in the amount of cellular p68
kinase. The steady-state amount of p68 kinase was found
to be reduced in cells stably expressing Tat. Thus, the
potential translational inhibitory effects of the TAR RNA
region, mediated by activation of p68 kinase, may be
down-regulated by Tat during activation of the latent
virus [ 22 ] . Along these lines, a Tat peptide
antagonist, which bound specifically to TAR RNA and
competed with Tat for binding, reduced Tat-dependent
translation [ 42 ] .
Finally, upregulation of translation genes in Tat
expressing cells is specially intriguing in light of the
recent discovery of internal ribosome entry sites (IRESs)
in HIV-1 gag ORF [ 43 ] . IRESs are thought to promote
initiation of translation by directly binding to
ribosomes, in a manner independent of the mRNA cap or of
scanning through upstream sequences. Since, the TAR is
located at the 5' end of all HIV-1 RNA transcripts and
the presence of secondary structure at or near the 5' end
of RNAs reduces the accessibility of the 5' cap to eIF4F,
it is thought that this feature of HIV-1 mRNAs can
inhibit their cap-dependent translation [ 44 45 46 ] .
Therefore, a possible function of the HIV-1 gag IRES
might be to serve as a mechanism to bypass the structural
barriers to cap-dependent translation by recruiting
ribosomes easily and directly to the gag ORFs. IRES
entirely contained within a translated ORF has been shown
in the MMLV gag [ 47 ] , and host mRNA encoding p110
PITSLREand p58 PITSLRE [ 48 ] . Along these lines,
cap-dependent translation may be cell cycle regulated,
especially when cells are arrested at the G2 phase of the
cell cycle, where the cap-dependent translation of most
cellular host cell mRNAs is inhibited [ 49 50 51 ] .
Modulation of signal transduction pathway
Results in Table 3indicate that many seemingly
different pathways are being regulated by Tat. However,
the signal transduction pathway, MAPK, has been shown to
control and be upstream of DNA-replication,
transcription, and cell cycle pathways [ 52 53 54 ] . The
mitogen-activated protein kinase (MAPK) pathway,
consisting of the MAP kinase kinases (MKKs) 1 and 2, and
extracellular signal-regulated kinases (ERKs) 1 and 2,
which have been implicated in diverse cellular processes
including proliferation, transformation, and cell
differentiation [ 53 ] . The MAP kinase (MAPK) pathway
has emerged as a crucial route between membrane-bound Ras
and the nucleus. This MAPK pathway encompasses a cascade
of phosphorylation events involving three key kinases,
namely Raf, MEK (MAP kinase kinase) and ERK (MAP kinase).
The MAPK pathway controls ERKs 1 and 2, c-Jun N-terminal
kinase (JNK), and p38. These signaling pathways in turn,
activate a variety of transcription factors including
NF-kappaB (p50/p65), AP-1 (c-Fos/c-Jun), and CREB
phosphorylation, which in turn coordinate the induction
of many genes encoding inflammatory mediators.
Cytokine receptors such as IL-3, GM-CSF, and the
interferons transmit their regulatory signals primarily
by the receptor-associated Jak family of tyrosine
kinases, and activate STAT transcription factors.
Activated STAT5 proteins are detected in reduced levels
in lymphocytes recovered from HIV-infected patients and
immunocompromised mice. Both of these types of receptor
signaling pathways have recently been shown to interact
with serine/threonine kinases such as MAP kinases. A
common intermediate pathway initiating from receptors to
the nucleus is the Ras/Raf/MEK/ERK (MAPK) cascade, which
can result in the phosphorylation and activation of
additional downstream kinases and transcription factors
such as p90Rsk, CREB, Elk, and Egr-1 [ 55 56 ] .
Therefore, it is intriguing that Tat expressing cells
show down-regulation of MAPK components (Table 3, Figure
3Band 3C), essential mediators between receptors and
nuclear transcription factors. This would imply that
latently infected cells that express Tat (doubly spliced
RNA) and not the whole virus (all three classes of the
RNA), can control signal transduction related to membrane
and transcriptional signaling (Figure 6).
Interestingly, Tat, through the RGD motif, which
controls integrin-based cell signaling, has been reported
to mediate the activity of phosphotyrosine
phosphatase(s). This in turn which would lead to a
decrease in the levels of phosphotyrosine-containing
proteins such as ERK-2/p42MAPK kinases [ 57 ] .
Cysteine-rich and basic Tat peptides have been shown to
inhibit VEGF-induced ERK activation and mitogenesis.
These peptides also inhibited proliferation,
angiogenesis, and ERK activation induced by basic
fibroblast growth factor with similar potency and
efficacy [ 58 ] . Consistent with this model, it has been
shown that treatment of neural cells with culture
supernatants from HAART-treated subjects, which
presumably contain extracellular Tat, resulted in
down-regulation of the JNK, AKT, and ERK kinases [ 59 ]
.
Finally, activation of MAPKs has been shown to
activate the singly spliced and unspliced (genomic)
latent HIV-1 virus. For instance, the signal transduction
pathways that regulate the switch from latent to
productive infection have been linked to MAPK. The
induction of latent HIV-1 expression has been shown to be
inhibited by PD98059 and U0126, specific inhibitors of
MAPK activation. The MAPK acts by stimulating AP-1 and a
subsequent physical and functional interaction of AP-1
with NF-κB, resulting in a complex that synergistically
transactivates the HIV-1 [ 60 ] . At the level of
infection and entry, the activation of MAPK through the
Ras/Raf/MEK (MAPK kinase) signaling pathway enhances the
infectivity of HIV-1 virions. Virus infectivity can be
enhanced by treatment of cells with MAPK stimulators,
such as serum and phorbol myristate acetate, as well as
by coexpression of constitutively activated Ras, Raf, or
MEK in the absence of extracellular stimulation [ 61 ] .
Also, following infection, efficient disengagement of the
reverse transcription complex from the cell membrane and
subsequent nuclear translocation, requires
phosphorylation of the reverse transcription complex
components by ERK/MAPK; demonstrating a critical
regulation of an early step in HIV-1 infection by the
host cell MAPK signal transduction pathway [ 62 ] .
Therefore, Tat down-regulation of the MAPK pathway in
latent cells implies that much of the host signal
transductions connected to activation are down-regulated,
and at the same time, these cells may be refractory to
subsequent infection by other viruses.
Thymosin family members, and cell cycle
Prothymosin α (ProTα) belongs to the α-Thymosin family
which comprises different polypeptides widely distributed
within animal tissues. Although its role has remained
controversial, it is involved in the increase of
immediate early genes such as c-myc [ 63 ] , which is
upstream of cyclin D synthesis and necessary for cell
division [ 64 ] . In humans, ProTα is coded by a gene
family of six members. One of them contains introns,
exons and classic regulatory signals, while the remaining
five are intronless genes [ 59 ] located on chromosome 2
[ 66 ] . There are two mRNA transcripts, which arise in a
ratio of 9:1 (shorter/longer form), where only the long
transcript is regulated by extracellular signals.
It has been demonstrated that malignant tissues with
accelerated cell cycle show higher levels of ProTα
expression than normal or surrounding healthy tissues [
67 ] . ProTα was shown as a marker for breast cancer [ 68
] , hepatocarcinoma [ 69 ] , and plasma levels of its
derivative Tα
1 been proposed as a marker for the
prognosis of lung cancer [ 70 ] . In ligand blotting
assays, ProTα bound only to chromatin pools and nuclear
fractions where histone H1 was present [ 71 72 ] . The
analysis of the interaction of ProTα with H1-containing
chromatin suggests a putative role for ProTα in the
fine-tuning of the stoichiometry and/or mode of
interaction of H1 with chromatin [ 73 ] . Interestingly,
HL-60 cells overexpressing ProTα show an enhancement of
accessibility of micrococcal nuclease to chromatin,
implying relaxed chromatin structure for enhanced cell
cycle gene expression [ 74 ] .
A broad study using several mononuclear and
fibroblastic cell lines has shown that ProTα mRNA
accumulation is cell cycle phase-dependent. In the U937
monocytic cell line, ProTα mRNA peaked at the end of S/G2
phase and fell towards the entry into the new G1 phase.
More prominent mRNA regulation was found in the
fibroblastic cell lines CV1 and NIH3T3, with peak mRNA
levels at the end of S-phase. In all cases the expression
pattern coincided with that of cyclin B and Cdc2/cyclin B
activation [ 75 ] .
It is interesting to note that Cdc2 (Acc# X05360),
Cdc10 homolog (Acc# S72008), and Cdc37 (Acc# U43077) were
all up-regulated in Tat expressing cells. Cdc2, a
catalytic subunit of cyclin-dependent kinases, is
required for both the G1-to-S and G2-to-M transitions. In
the fission yeast Schizosaccharomyces pombe, the
execution of Start requires the activity of the Cdc2
protein kinase and the Cdc10/Sct1 transcription complex.
The loss of any of these genes leads to G1 arrest [ 69 ]
.
Cdc37 encodes a 50-kDa protein that targets
intrinsically unstable oncoprotein kinases including
Cdk4, Raf-1, and v-src to the molecular chaperone Hsp90,
an interaction that is thought to be important for the
establishment of signaling pathways. Cdc37 expression may
not only be required to support proliferation in cells
that are developmentally programmed to proliferate, but
may also be required in cells that are inappropriately
induced to initiate proliferation by oncogenes. For
instance, MMTV-Cdc37 transgenic mice develop mammary
gland tumors at a rate comparable to that observed
previously in MMTV-cyclin D1 mice, indicating that Cdc37
can function as an oncogene in mice and suggests that the
establishment of protein kinase pathways mediated by
Cdc37-Hsp90 can be a rate-limiting event in
transformation [ 76 ] . Also, analysis of proteins that
co-immunoprecipitated with Cdk6 and Cdk4 has shown
complexes containing both Hsp90 and Cdc37 [ 77 78 79 ]
.
Cdc37 also promotes the production of Cak1. Cak1 in
yeast is the human homolog of CAK trimeric enzyme
containing CDK7, cyclin H, and MAT1. Both human and yeast
Caks function as RNA polymerase II CTD kinase, Cdk
activating kinase, and DNA damage/repair enzymes. Cdc37,
like its higher eukaryotic homologs, promotes the
physical integrity of multiple protein kinases, perhaps
by virtue of a cotranslational role in protein folding [
80 ] . Finally, Hsp90/Cdc37 has recently been shown in
the stabilization/folding of Cdk9 as well as the assembly
of an active Cdk9/cyclin T1 complex responsible for
P-TEFb-mediated Tat transactivation [ 81 ] .
Transcription and chromatin remodeling
factors
A highly ordered chromatin structure presents a
physical obstacle for gene transcription; presumably by
limiting the access of transcription factors and RNA
polymerase II core machinery to target DNA [ 82 83 ] . In
concert with the observation that corepressors are
associated with HDAC activities [ 84 85 ] , it appears
that the transcriptional outcome of nuclear receptors is
determined by the balance of histone acetylation and
deacetylation activities, and that ligands serve as a
switch to recruit HATs with the concomitant dismissal of
HDACs. Signal transduction pathways add another layer of
regulation to the functions of CBP/p300. In the case of
the POU homeodomain factor Pit-1, transcriptional
activity is potentiated by MAPK pathways [ 86 ] .
Therefore, down-regulation of MAPK pathway members in Tat
expressing cells, as seen in Table 3, is consistent with
decreased phosphorylation of DNA binding factors such as
Pit-1, and overall lower DNA binding activity. Here, we
describe the effect of coactivator proteins SRC-1 (Acc#
AJ000882, U90661, Table 1) and p300 (Acc# U01877, Table
3), and their relation to differentiation genes such as
retinoic acid receptor (RAR/PML, Acc#: X06614, Table 1),
and Leptin receptor variant (Acc#: U66496, Table 1); all
of which are down-regulated in Tat expressing cells
(Figure 5).
Over the past three decades a great deal of evidence
has accumulated in favor of the hypothesis that steroid
receptor hormones act via regulation of gene expression.
The action is mediated by specific nuclear receptor
proteins, which belong to a superfamily of
ligand-modulated transcription factors that regulate
homeostasis, reproduction, development, and
differentiation [ 87 ] . This family includes receptors
for steroid hormones, thyroid hormones, hormonal forms of
vitamin A and D, peroxisomal activators, and ecdysone [
88 ] . Nuclear hormone receptors are ligand-dependent
transcription factors that regulate genes critical to
such biological processes as development, reproduction,
and homeostasis. Interestingly, these receptors can
function as molecular switches, alternating between
states of transcriptional repression and activation,
depending on the absence or presence of a cognate
hormone, respectively. In the absence of cognate hormone,
several nuclear receptors actively repress transcription
of target genes via interactions with the nuclear
receptor corepressors SMRT and NCoR. Upon binding of the
hormone, these corepressors dissociate from the DNA-bound
receptor, which subsequently recruits a nuclear receptor
coactivator (NCoA) complex. Prominent among these
coactivators is the SRC (steroid receptor coactivator)
family, which consists of SRC-1, TIF2/GRIP1, and
RAC3/ACTR/pCIP/AIB-1. These cofactors interact with
nuclear receptors in a ligand-dependent manner and
enhance transcriptional activation via histone
acetylation/methylation and recruitment of additional
cofactors such as CBP/p300 [ 89 ] . CBP/p300 has been
implicated in the functions of a large number of
regulated transcription factors based primarily on
physical interaction and the ability to potentiate
transcription when overexpressed [ 90 ] . In the case of
nuclear receptors, the interaction with CBP/p300 is
ligand-dependent and relies on the conserved nuclear
receptor functional domain, AF-2 (activation function 2).
In vivo studies have supported the
conclusion that CBP/p300 are components of the
hormonal-regulation of transcription in fibroblasts
isolated from a p300-/- mouse; and loss of the p300 gene
severely affects retinoic acid (RA)-dependent
transcription [ 91 ] . In a separate study using
hammerhead ribozymes that specifically cleave CBP or p300
mRNA, Kawasaki et al [ 92 ] reported that reduced
cellular CBP or p300 levels resulted in compromised
expression of endogenous RA-inducible genes such as
p21/Waf1 and p27 cdk inhibitors. Along this line, Tat
expressing cells have lower levels of p21/Waf1 presumably
due to inactivation of p53 and a lack of p300/RA- induced
gene expression. Consistent with this interpretation, CBP
and p300 harbor transcriptional activation of
ligand-induced RA or ER function on a chromatinized
template [ 93 ] .
The NcoA family members constitute SRC-1/NcoA-1 [ 89 ]
, TIF2/GRIP1/NcoA-2, [ 94 95 ] and pCIP/ACTR/AIB1 [ 96 97
98 ] proteins, which interact with liganded RA receptor
(RAR), and CBP/p300. Overexpression of these NCoA factors
enhances ligand-induced transactivation of several
nuclear receptors [ 99 ] . A weak intrinsic HAT activity
has been reported in SRC-1/NCoA-1 and pCIP/ACTR/AIB1,
suggesting that chromatin remodeling may also be a
function of these NCoA factors [ 99 100 ] ; although they
do not appear to contain regions homologous to the HAT
domains of CBP/p300 or p/CAF. Structure-function analysis
of the NCoAs have revealed multiple copies of a signature
motif, LXXLL, with conserved spacing that is required for
interaction with nuclear receptors and CBP/p300 [ 99 101
] . Intriguingly, different LXXLL motifs are required for
PPARγ (Peroxisome Proliferator activated receptor γ, a
gene down-regulated in Tat expressing cells; Acc# L07592,
Table 1) function in response to different classes of
ligands, suggesting distinct configuration of assembled
complexes.
Taken together, through the use of microarray
technology, we have described one of the first
observations about how Tat is able to control various
host cellular machineries. Although our data is
consistent with most of the cited literature on the
effects of Tat in infected host and uninfected bystander
cells, we caution that the transcriptional profiling in
chronically infected cells such as ACH2 or H9/Tat cells
may not necessarily be representative of the pattern of
expression observed in most cells infected by other group
M, N, or O HIV-1 isolates.
We recently extended our observations by utilizing
other HIV-1 infected cells which normally express Tat
(U1), and addition of exogenous purified Tat to
uninfected PBMCs. Preliminary results using western blots
supports the idea that genes which were altered in H9/Tat
system also showed a similar level of change in few of
the tested genes (Figure 3C). This notion of consistency
was further confirmed using the IL-8 activation by Tat.
Interleukin-8 (IL-8) belongs to the CXC chemokine family
and is secreted by several different cell types,
including monocytes, neutrophils, endothelial cells,
fibroblasts, and T lymphocytes. IL-8 production (induced
by several stimuli, including IL-1, TNF-, and phorbol
myristate acetate) is primarily regulated at the
transcriptional level. IL-8 is a potent chemotactic
factor for granulocytes and T lymphocytes, and is found
in HIV-infected individuals. The CXC chemokine IL-8 does
not bind to CCR5. It has previously been shown that IL-8
mRNA induction was seen less then 1 h after Tat (72aa)
stimulation, and levels remained elevated for up to 24 h,
leading to IL-8 protein production [ 102 ] . Along these
lines, we have previously shown that the IL-8 gene is
expressed in a cell cycle-dependent manner in cells that
express the Tat protein, and the induction is during the
S phase of the cell cycle and regulated by stable NF-kB
binding to the IL-8 promoter [ 103 ] . When looking for
IL-8 at the G1/S border, we found that all Tat containing
cells, including PBMCs that were treated with exogenous
Tat showed an up-regulation of IL-8 in the supernatant
(Figure 4), further implying that results obtained from
the H9/Tat system may infact be of general physiological
relevance in vivo.
Finally, throughout the current study we came across
some technical findings that were critical in the
confirmation of most of our results. For instance, few
genes did not correlate in their activation or
suppression levels when comparing fold changes between
microarrays and protein levels using western blot
analysis. We suspect this is because many genes that are
transcribed may not necessarily be translated, due to
their cell cycle stage, 5' stem and loop RNA structures,
varying half-lives of proteins and mRNAs, and a host of
other unknown variables. Also, specific changes that
occur in a cell may not be required in redundant pathways
that score for a specific function. This is commonly seen
in the differences between HIV-1 infected or Tat
expressing
in vitro cell lines and AIDS
patients PBMC samples. Therefore, other microarrays would
have to be performed on purified infected PBMCs to
confirm most of the changes observed in Tables 1, 2, and
3. Unfortunately, to date this particular issue is not
feasibly addressable, since it is not possible to isolate
a homogenous population of infected T- or Monocytic cells
from AIDS patients. Also, confirmatory tests for protein
expression would have to be done with both hydrophilic
and hydrophobic extraction buffers. For instance, we have
observed that PCNA protein, which is up-regulated in Tat
expressing cells, extract best with hydrophobic buffers
from the nucleus, presumably due to its binding to DNA
replication machinery (data not shown). Future
experiments will address issues related to differences
between various HIV-1 Tat clades, host expression levels
between T- and Monocytic cells, and its effect at various
stages of the cell cycle.
Conclusions
Expression profiling from HIV-1 or Tat expressing cells
holds great promise for rapid functional analysis. Here, we
have described the effect of Tat and its alterations with
the host cellular gene expression. We observed that more
than 2/3 of the cellular genes tested were down-regulated
by Tat. These genes belong to receptor, co-receptor, and
co-activator pathways that are part of serine/threonine
receptor tyrosine kinase, Ras/Raf/MEK/ERK (MAPK) cascade,
which control proliferative and/or differentiation signals.
We also observed a great deal of increase in the host cell
translation apparatus with the possible aim of optimizing
viral mRNA translation prior to viral maturation and
release. Therefore, HIV-1 accessory doubly spliced messages
such as Tat, may control the host gene expression in
latently infected cells, and determine not only viral
transcription, but also the fate of post-transcriptional
events.
Materials and method
Cell culture
ACH
2 cells are HIV-1 infected CD4
lymphocytic cells, with an integrated wild-type
single-copy chromatinized DNA. The CEM T cell (12D7) is
the parental cell for ACH
2 cells. ACH2 cell lines has a single
copy of LAI strain proviral sequence. The TAR has a point
mutation at (C37 -> T), which no longer responds
(efficiently) to Tat. However, the cell line is fully
capable of making infectious virus in presence of TNF,
PHA, PMA, and a host of other stimuli. H9 and H9/Tat
cells are both CD4+ Lymphocytic cells, where H9 cells
carry a control integrated vector without the Tat open
reading frame, and H9/Tat cells carry integrated Tat
expression vector. Both cell lines were a generous gift
of George Pavlakis (NCI, NIH). U1 is a monocytic clone
harboring two copies of the viral genome from parental
U973 cells. All cells were cultured at 37°C up to 10
5cells per ml in RPMI-1640 media, containing 10% Fetal
Bovine Serum (FBS) treated with a mixture of 1%
streptomycin and penicillin antibiotics, and 1%
L-glutamine (Gibco/BRL). Phytohemagglutinin-activated
PBMC were kept in culture for 2 days prior to addition of
Tat protein. Isolation and treatment of PBMC were
performed by following the guidelines of the Centers for
Disease Control. Approximately 5 × 10 6PBMC were used for
treatment of wild type and K41A Tat mutant (100 ng/ml)
proteins. After an initial incubation for one hr with Tat
proteins, cells were washed and cultured in complete
media for 24 hrs, prior to western blots. pCEP4, eTat
cells were HeLa cells stably transfected with either a
backbone control plasmid (pCEP4; Invitrogen) or a plasmid
expressing Tat (1-86) with a C-terminal epitope tag
(eTat) [ 103 ] . HeLa cell lines containing either the
control or eTat plasmid were selected by single-cell
dilution. Both cell types were selected and maintained
under 200 μg of hygromycin per ml. Verification of Tat
transcriptional activity was achieved by electroporation
of reporter plasmids as previously described [ 103 ]
.
Cell cycle analysis
Hela cells were blocked with hydroxyurea (Hu) (2 mM)
for 14 h. Following the block, cells were released by
being washed twice with phosphate-buffered saline (PBS)
and by the addition of complete medium. All suspension
cells were treated with 1% serum for 48 hrs prior to
addition of Hu. Supernatants were collected and analyzed
by an IL-8 ELISA according to the manufacturer's
instructions (Biosource International). For controls,
each sample, approximately 1 × 10 6cells was processed
for cell sorting. Cells were washed with PBS and fixed by
addition of 500 μl of 70% ethanol. For
fluorescence-activated cell sorting (FACS) analysis,
cells were stained with a cocktail of propidium iodide
(PI) buffer (PBS with Ca2+ and Mg2+, RNase A [10 μg/ml],
NP-40 [0.1%], and PI [50 μg/ml]) followed by cell-sorting
analysis. FACS data acquired were analyzed by ModFit LT
software (Verity Software House, Inc.).
Cell extract preparation and immunoblotting
All cells were cultured to mid-log phase of growth,
washed with PBS without Ca 2+and Mg 2+, and lysed in a
buffer containing 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5
mM EDTA, 50 mM NaF, 0.2 mM Na
3 VO
4 , 1 mM DTT, 0.5% NP-40 and protease
inhibitors (Protease inhibitor cocktail tablets,
Boehringer Mannheim, one tablet per 50 ml). The lysate
was incubated on ice for 15 min, and microcentrifuged at
4°C for 10 min. Total cellular protein was separated on
4-20% Tris-glycine gels (Novex, Inc.) and transferred to
a polvinylidene difluoride (PVDF) membranes (Immobilon-P
transfer membranes; Millipore Corp.) overnight at 0.08 A.
Following the transfer, blots were blocked with 5%
non-fat dry milk in 50 ml of TNE 50 (100 mM Tris-Cl [pH
8.0], 50 mM NaCl, 1 mM EDTA) plus 0.1% NP-40. Membranes
were probed with a 1:200-1:1000 dilution of antibodies at
4°C overnight, followed by three washes with TNE 50 plus
0.1% NP-40. All antibodies used in this study were
purchased from Santa Cruz Biotechnology. The next day,
blots were incubated with 10 ml of 125I-protein G
(Amersham, 50 μl/10 ml solution) in TNE 50 plus 0.1%
NP-40 for 2 hrs at 4°C. Finally, blots were washed twice
in TNE 50 plus 0.1% NP-40 and placed on a PhosphorImager
cassette for further analysis.
Total RNA purification
Cells were grown to mid-log phase of growth (5.0 × 10
6), pelleted, and washed twice with cold D-PBS without Ca
2+/Mg 2+. Total RNA was extracted on ice using Trizol
Reagent (Life Technologies, Inc.). Purified RNA was then
analyzed on a 1% agarose gel for quality and quantity
prior to each experiment.
Glass slide microarray
Gene expression analysis was performed using
Micromax™: Human cDNA Microarray System I (cat# MPS101,
NEN Life Science Products). On a glass microarray slide,
2400 know human genes were arrayed into 4 separate grids
(A, B, C, D), containing 600 genes each (gene description
and location on microarrays available at NEN website:
www.nenlifesci.com). All human genes were ~2200 bp cDNAs,
and were characterized from 50+ human cDNA libraries
(AlphaGene, Inc., Woburn, MA). In addition to the human
genes, three plant control genes were spotted on each
grid and were utilized to balance the Cyanine-3 (Cy-3)
and Cyanine-5 (Cy-5) fluorescence signals.
A total of 8 μg each of H9 (control sample) and H9/Tat
(test sample) mRNAs were reverse transcribed into Biotin
and Dinitrophenyl (DNP) labeled cDNA, respectively. After
cDNA quality and quantities were analyzed, both cDNAs
were then pooled and simultaneously hybridized overnight
at 65°C onto the glass microarray. The next day, the
microarray slide was serially washed in 0.5× SSC (Sodium
Citrate-Sodium Chloride) + 0.01% SDS (Sodium Dodecyl
Sulfate), 0.06× SSC + 0.01% SDS, and 0.06× SSC. Next, the
Tyramide Signal Amplification (TSA™) was then used to
amplify the Cy-3 and Cy-5 signals using antibody-enzyme
conjugates, α-DNP-Horseradish peroxidase (HRP) and
α-Streptavidin-HRP with Tyramide linked Cy-3 and Cy-5.
Screening and data analysis was performed by NEN.
cDNA filter hybridization
Gene expression of CEM and ACH2 were performed using
Atlas Human cDNA Expression Array (Clontech Laboratories
Inc., Palo Alto, CA) according to the manufacturer's
directions. One μg of poly A +RNA each was DNase I
treated, purified using a CHROMA SPIN-200 column, and
reverse transcribed into 32P-labeled cDNA. The CHROMA
SPIN-200 column was used to purify the 32P-labeled cDNA
from unincorporated 32P-labeled dNTPs and small (<0.1
kb) cDNA fragments. Each sample was then hybridized to a
human cDNA expression array overnight with continuous
agitation at 68°C. The next day, the array was washed
three times with gentle agitation, first wash with 2× SSC
+ 1% SDS and the last two washes with 0.1× SSC + 0.5% SDS
at 37°C. Array was exposed to a PhosphorImager Cassette
and analyzed using ImageQuant software.
Northern blots
Total cellular RNA was extracted using the RNAzol
reagent (Gibco/BRL). Total RNA (20 μg) was isolated from
various cells and ran on a 1% formaldehyde-agarose gel
overnight at 75 V, transferred onto a 0.2 μm
nitrocellulose membrane (Millipore Inc.), UV
cross-linked, and hybridized overnight at 42°C with
32P-end-labeled 40 mer oligo probes including p21/Waf1,
C-myc, Pro-thymosin, Actin, Tat, and Ubiquitin
(Loftstrand, Gaithersburg, Md.). Next day, membranes were
washed two times for 15 min each, with 10 ml of 0.2%
SDS-2XSSC at 37°C, exposed, and counted on PhosphorImager
Cassette.
Viral infection and ELISA assay
Both H9 and H9/Tat cells were infected in the presence
of 10 ug of polybrene. For PBMC infections, PHA activated
PBMCs were kept in culture for 2 days prior to each
infection. Isolation and treatment of PBMCs were
performed by following guidelines from the CDC
(Isolation, culture, and identification of HIV,
Procedural Guide, July 1991, Atlanta, GA). Approximately
2 × 10 6of H9 or H9/Tat cells, and 5 × 10 6PBMC cells
were infected with either an HXB-2 (CXCR4, T-tropic), or
BaL (CCR5, Macrophage-tropic) at 5 ng of p24 gag antigen/
HIV-1 strain. Both viral isolates were obtained from the
NIH AIDS research and reference reagent program. After 8
hrs of infection, cells were washed and fresh media was
added. Samples were collected every 3 rdday and stored at
-20 C for p24 gag ELISA. Media from HIV-1 infected cells
were centrifuged to pellet the cells and supernatants
were collected, and diluted to 1:100 to 1:1000 in RPMI
1640 prior to ELISA. The p24 gag antigen level was
analyzed by HIVAG™-1 Monoclonal antibody Kit (Abbott
Laboratories, Diagnostics Division).
Authors' Contributions
CF, and FS carried out the ACH2 and H9/Tat microarrays.
LD, CE, IZ, CGL, and KW aided in westerns, northerns, p24
and ELISA assays. AM, KK, SB, AP, and FK aided in data
interpretation, Bioinformatics, literature searches and
writing the manuscript.
