Heat Shock Factor 1 Mediates Latent HIV Reactivation
Heat Shock Factor 1 Mediates Latent HIV Reactivation
Xiao-Yan Pan 2
Wei Zhao 1 2
Xiao-Yun Zeng 2
Jian Lin 2
Min-Min Li 0
Xin-Tian Shen 2
Shu-Wen Liu 1 2
0 Center for Clinical Laboratory , First
1 State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Southern Medical University , Guangzhou , China
2 Guangdong Provincial Key Laboratory of Drug Screening, School of Pharmaceutical Sciences, Southern Medical University , Guangzhou 510515 , China
OPEN HSF1, a conserved heat shock factor, has emerged as a key regulator of mammalian transcription in response to cellular metabolic status and stress. To our knowledge, it is not known whether HSF1 regulates viral transcription, particularly HIV-1 and its latent form. Here we reveal that HSF1 extensively participates in HIV transcription and is critical for HIV latent reactivation. Mode of action studies demonstrated that HSF1 binds to the HIV 5′-LTR to reactivate viral transcription and recruits a family of closely related multi-subunit complexes, including p300 and p-TEFb. And HSF1 recruits p300 for self-acetylation is also a committed step. The knockout of HSF1 impaired HIV transcription, whereas the conditional over-expression of HSF1 improved that. These findings demonstrate that HSF1 positively regulates the transcription of latent HIV, suggesting that it might be an important target for different therapeutic strategies aimed at a cure for HIV/AIDS.
The long-lived latent viral reservoir of HIV-1 prevents its eradication and the development of a cure1. The recent
combination antiretroviral therapy (cART) aimed at inhibiting viral enzymatic activities prevents HIV-1
replication and halts the viral destruction of the host immune system2. However, proviruses in the latent reservoir
persist in a transcriptionally inactive state, are insuppressible by cART and undetectable by the immune system3.
Proviruses in the latent reservoir primarily contribute to the rapid rebound of viremia after the cessation of
cART4. Purging this reservoir is important for the development of a cure for AIDS and requires an understanding
of latency mechanisms5–7.
In recent years, several host cell proteins and viral regulatory proteins that influence retroviral latency have
been identified. The respective lack of cellular transcription factors and HIV-1 Tat protein limits the initiation and
elongation of viral transcription in resting CD4+ T cells8. Genome-wide siRNA screenings have identified a total
of 842 host genes associated with HIV-1 infections9, and a genome-wide mass spectrometry-based proteomic
analysis has revealed 497 HIV-human protein-protein interactions involving 435 individual human proteins in
HEK293 and Jurkat cells10. Notably, only a small fraction of cellular factors influences the latency maintenance
and reactivation stages; thus, it is likely that additional cellular factors exploited by the virus during these stages
remain to be identified. These factors might be engaged during transcriptional initiation and subsequent
Until recently, cell stress proteins assisting latent reactivation, except for heat shock protein 90 (HSP90)11,
have scarcely been detected. Here, we present a previously underappreciated stress protein, heat shock factor
1 (HSF1)12, which extensively participates in latent HIV reactivation. First, we identified an important role for
HSF1 in latent reactivation under stress. Next, the binding of HSF1 to the HIV long terminal repeat promoter
(LTR) was detected during reactivation using chromatin immunoprecipitation (ChIP) assays, and two
accessory factors, p300 and positive transcription elongation factor b (p-TEFb), were confirmed to assist HSF1 in
maintaining function using co-immunoprecipitation (Co-IP) assays. Moreover, the acetylation of HSF1 was
also considered as key point to latent HIV reactivation. Definitively, knockout and overexpression HSF1 further
demonstrated its pivotal role in transcription. These data highlight an important role for HSF1 in HIV latency
reactivation, providing a deeper understanding of latency mechanisms. These findings suggest that HSF1 might
be a novel target for different therapeutic strategies against HIV/AIDS.
The cell stress response selectively reactivates HIV in latency cell models. To examine latent
HIV in response to various cellular stresses, a well established and widely used model of HIV latency based on
the Jurkat cell line, J-Lat 10.613 was adopted in the present study. Heat shock, innate immune stress, oxidative
stress and endoplasmic reticulum stress are four classical stresses that can be induced by hyperthermia, poly (I:C),
STA-4783 and thapsigargin respectively14. Stress responses were detected based on the phosphorylation of
eukaryotic initiation factor 2 alpha (p-eIF2α) in whole cells. As a result, J-Lat cells treated with these inducers showed
significant up-regulation of p-eIF2α expression in 12 hours (Fig. 1A). At the same time, poly (I:C) and STA-4783
increased the expression of GFP over five folds from the basal level, while thapsigargin upregulated the expression
of GFP slightly. Hyperthermia (39.5 °C) enhanced PHA-induced GFP expression which was in according with
the report by Roesch et al.15. Although these four inducers influenced latent HIV in different extent, these results
indicated that HIV is reactivated from latency in response to cell stress.
We next confirmed the relationship between HIV reactivation and p-eIF2α upregulation after treating cells
with a specific eIF2α dephosphorylase inhibitor (salubrinal), which effectively maintains and increases eIF2α
phosphorylation. As a result, salubrinal efficiently induced phosphorylation of eIF2α in a short time and
this effect was maintained by hyperthermia (Fig. 1D). Consistent with this, salubrinal reactivated latent HIV
in a dose-dependent manner and the reactivation was enhanced by hyperthermia (Fig. 1B). The upregulated
expression of the HIV capsid protein p24 was consistent with the percentage of GFP (Fig. 1D). Moreover, the
salubrinal-mediated reactivation of latent HIV appeared no toxicity to J-Lat 10.6 cells (Fig. 1C). As we observed,
salubrinal induced phosphorylation of eIF2α on HIV latency cell model J-Lat 10.6 in our research, which is in
agreement with the previous reports showing that it can induce eIF2α phosphorylation in other cell lines16–18. The
phosphorylation of eIF2α is a marker of stress19, while salubrinal can also inhibits ER stress. The possible
explanation is that salubrinal-induced phosphorylation of eIF2α triggers HSF1 playing role in cellular proteostasis thus
giving cell the ability to resist ER stress in return20,21.
In addition, ACH2 and U1 cell models, along with primary CD4+ T cells from HIV infected
individuals receiving suppressive ART were adopted to examine HIV transcription after treatment with salubrinal.
Consistently, the transcription of HIV genes such as gag, tat, vif and vpr quantitatively analyzed by real-time PCR
(RT-PCR) were efficiently upregulated after treatment with salubrinal for 24hours in the HIV latency cell models
ACH2, U1 and J-lat 10.6. Despite that PMA/Iono significantly upregulated the transcription of HIV 5-‘LTR and
there was no significance between control group and salubrinal treatment group based on data from three donors,
salubrinal could slightly upregulated the transcription of HIV 5′-LTR at 48 hour in primary CD4+ T cells from
suppressive HIV patients (Fig. 1E).
Interestingly, the latent HIV reactivation stimulated by salubrinal was not associated with global T cell
activation. The results on human peripheral blood mononuclear cells (PBMCs) displayed in Fig. 1F showed that
the expression of CD25 and CD69 was not up-regulated with salubrinal compared with prostratin treatment.
Additionally, the expression of CD25 and CD69 on J-Lat cells was slightly down-regulated following treatment
with hyperthermia (39.5 °C). Together, these results indicate that stress sensitizes latent HIV to reactivation across
cell models, and this is closely associated with the phosphorylation of eIF2α and the inhibition of global T cell
HSF1 is activated during HIV reactivation induced by stress inducer salubrinal. We hypothesized
that the cellular factors involved in the p-eIF2α downstream signaling pathway might play a major role in
reactivation. Therefore, mRNA expression of multiple cellular transcriptional factors (See supplemental Table S1) was
examined (Supplemental Figure S1). Indeed, the mRNA expression of AP-1 complex subunits, NFAT and ATF
family members and HSF1 were markedly up-regulated. This effect was even more obvious after co-treatment
with hyperthermia, and the same tendency was observed in Fig. 2B. As we observed, the gene expression of
c-fos, c-Jun and ATF3 were significantly upregulated within 24 hour in a time dependent manner under the
treatment with salubrinal at 39.5 °C. In comparison, the gene expression of NFAT2 and HSF1 changed slightly. Yet,
the nuclear import of transcription factors were examined by Western blotting, and phosphorylated HSF1
dramatically accumulated in the nucleus when cells were treated with salubrinal for 2 hours and returned to basal
levels after 4 hours (Fig. 2A). The changes in c-fos and ATF3 were consistent with phosphorylated HSF1, and the
increased level of NFATc1 remained unchanged in 4 hours. However, RelA which was notably induced by TNF-α,
PMA and prostratin22, was not up-regulated after salubrinal treatment.
Next, cells treated with salubrinal or the PKC pathway modulator prostratin were respectively co-treated with
AP-1 inhibitor (parthenolide, PTN), NFATc1 inhibitor (cyclosporine A, CsA), NF-κB inhibitor (bay 11–7082)
or HSF1 –p-TEFb complex inhibitor (KRIBB11). AP-1 and NFATc1 inhibition impaired reactivation whether
induced by prostratin or salubrinal, and discrepancy was observed after NF-κB inhibition. The expression of
GFP was reduced in J-Lat cells when bay 11–7082 was co-treated with prostratin but persisted when co-treated
with salubrinal for 48 hour (Fig. 2C,D). Interestingly, HSF1 inhibition dramatically reduced latent reactivation in
a dose-dependent manner, regardless of the reactivation was induced by salubrinal or prostratin (Fig. 2E). These
results suggest that multiple transcription factors are involved in latent virus reactivation. As HSF1 and its active
form phosphorylated HSF1 (Ser320) showed up a significant increase tendency in the nucleus and GFP% was
reduced by KRIBB11 treatments, we deduced that HSF1 may play a role in HIV latency reactivation in addition
to the known transcription factors AP-1 and NFATc1. These results also suggest that the latent HIV reactivation
induced by salubrinal is HSF1-dependent and NF-κB-independent.
HSF1 mediates the participation of Heat Shock Proteins (HSPs) in HIV latency reactivation.
HSF1 is a host transcription factor for HSPs and other stress response genes under stresses23, we simply
speculated that HSF1 downstream factor may be involved in HIV latency reactivation. The transcription of HSP genes
was detected by RT-PCR. As showed in Fig. 3A, the mRNA expression of HSP90, HSP70, HSP27 and HSF1 were
up-regulated in J-Lat cells under stimulation with different temperature such as 39.5 °C and 42 °C, and
hyperthermia could efficiently enhanced the expression of HSF1 and its downstream genes. Meanwhile, the significant
up-regulation of HSP90, HSP70 and HSP27 mRNA expression were observed after treatment with prostratin and
salubrinal (Fig. 3A).
To explore whether HSP90, HSP70 and HSP27 influence HIV reactivation, we used AUY922 and 17-DMAG,
Ver-155008, and MK-2 inhibitor to inhibit HSP90, HSP70, and HSP27, respectively. Consistent with the results
from Anderson et al.11, HSP90 positively participated in latency reactivation, and the inhibition of HSP90 by
AUY922 and 17-DMAG reduced GFP expression in a dose-dependent manner (Fig. 3B). Similar to HSP90,
HSP70 inhibition by Ver-155008 also impaired latent HIV reactivation. These two HSPs are positively involved in
latent reactivation under the experimental conditions used in the present study. Additionally, the HSP70
activator YM-08 slightly increased GFP% in a dose-dependent manner (Fig. 3C) confirmed the positive role of HSP70
in HIV latency reactivation. In contrast, we presented that HSP27 negatively affected latent reactivation, due to
that the inhibition of HSP27 by MK2 inhibitor induced GFP expression rather than reduced that (Fig. 3C). These
results implied that HSPs actively express during HIV transcription whether induced by stress inducer salubrinal
or non-stress inducer prostratin, and further potentiate our finding that HSF1 plays an important role in latent
HSF1 recruiting p300 for self-acetylation is a committed step for HIV latency reversal. In addi
tion to p-TEFb, p300 has been detected involving in the complex recruited by HSF1. In Fig. 5A, the binding of
p300 on HSF1 was increased under salubrinal treatment at 2 hour, and impaired by p300 inhibitor C646. At
the same time, the acetylation of HSF1 was significantly increased in the present of salubrinal and decreased
by C646 (Fig. 5B) which was demonstrated by IP experiments with polyclonal acetylated lysine antibody. As a
result, the acetylation of HSF1 and the recruitment of acetylase p300 by HSF1 synchronized in the process of HIV
reactivation under treatment with salubrinal. Now that the acetylation of HSF1 is happened in the reactivation
process, we verified its necessity through inhibiting p300 by C646, and the results is predictable that p300
inhibition impaired latent HIV reactivation induced salubrinal. Similarly, latent HIV reactivation induced salubrinal
was inhibited by C646 in a dose-dependent manner (Fig. 5D).
Based on the result that HSF1 recruits p300 to acetylate itself, we speculated that HSF1 acetylation may
extensively participates in latency reactivation. On the basis of the report that p300, HDAC, NAD-dependent
deacetylase Sirtuin-1 (Sirt1) and proteasome contribute to acetylation of HSF126, we validated p300 inhibitor
C646 and HDAC inhibitor SAHA, Sirt1 activator resveratrol and inhibitor EX527, proteasome-ubiquitin
inhibitors MG-132 and hemin on J-Lat cells as Fig. 5C indicated. It was observed that SAHA, resveratrol, MG132 and
hemin which have been reported to reactivate latent HIV from other pathways, exerted strong ability of latent
HIV reactivation in our research, too. SAHA reactivated latent HIV (Fig. 5E) while C646 suppressed reactivation
induced by prostratin or salubrinal (Fig. 5D). Resveratrol promoted the activation of latent HIV while EX527 had
no effect on that (Fig. 5F). MG132 and hemin both boosted latent HIV reactivation (Fig. 5G). Despite these
compounds works on latent HIV reactivation on reported pathways, these results implied that they may also influence
latent HIV through acetylizing HSF1. Collectively, we summarized that HSF1 recruiting p300 for self-acetylation
is pivotal to the reactivation of latent HIV and the acetylation of HSF1 might extensively participate in latent HIV
reactivation, which might provide a better understanding of the mechanisms of HIV-1 latency reversal.
HSF1 broadly involves in latent HIV reactivation. To verify whether the requirement of HSF1 for
latent reactivation is universal, we co-treated latency reversing agents (LRAs) which reactivate latent HIV through
different signaling pathways (Supplemental Table S2) with KRIBB11 on J-Lat cells. As shown in Fig. 6A, HDAC
inhibitor SAHA, p-TEFb activators JQ1, PKC agonists prostratin, TNF-α, and dilazep, efficaciously up-regulated
GFP%. Meanwhile, the accumulation of phosphorylated HSF1 in the nucleus was markedly increased (Fig. 6B).
LRAs co-treated with KRIBB11 were employed to investigate the importance of HSF1 in latency reversal. Except
SAHA, and the latency reactivation induced by other four LRAs used in our study were all inhibited by KRIBB11
in a dose-dependent manner (Fig. 6C). As to the reason, one possible explanation was that SAHA might compete
with HSF1 for p-TEFb binding, resulting in latent reactivation enhancement. Despite that, we concluded that
HSF1 broadly involves in latent HIV reactivation.
Many cellular factors influence retroviral reactivation. NF-κB27, AP-128, NFAT29, Ets-130, p-TEFb and p300
directly interact with HIV 5′-LTR, extensively participating in gene transcription. HSF1 is another cellular factor
reported in the present study, which largely participates in reactivation in the presence or absence of stresses
(Fig. 6). Rawat et al. firstly demonstrated that HSF1 is induced during acute HIV-1 infection and positively
regulates HIV-1 gene expression through two distinct pathways31. Although HSF1 has been reported as a positive
regulator of HIV replication32, we examined the relationship between the modality and function of this factor
in latent HIV reactivation. Indeed, we propose that phosphorylation and acetylation are essential to the positive
potency of HSF1, while the preliminary and ubiquitylated forms of this protein manifest negative or no potency,
consistent with a previous report26.
Mono HSF1 is a repressor of HSP90 and HSP70 in the cytoplasm without any stimulators33, and mono HSF1
is transformed into a phosphorylated trimer then promote transcription under stress34. It then translocates into
the nucleus and binds to HSE32, followed by acetylation by p300 for gene activation and ubiquitylation by
ubiquitin for HSF1 degradation35. This mechanism might explain why the cotransfection of HSF1 with pNL4-3 does
not up-regulate the transcription of HIV, suggesting that the active form of HSF1 is necessary. Beyond that,
previous study showed that the mutant form of HSF1 (HSF1_ ϪRD) posseses transcriptional activity36. However,
the precise post-translational modifications controlling HSF1 function remain unclear, and additional studies
should focus on the phosphorylation, sumoylation, ubiquitination, and acetylation of HSF1 in novel primary
Besides that HSF1 is induced by stress, we demonstrate that HSF1 is universal factor in latent HIV
reactivation induced by LRAs and indispensable in HIV transcription. Particularly, the p-eIF2α-induced HSF1
activation reversed HIV latency was not associated with global T cell activation. Here we speculate that HSF1 is the
crucial factor mediating this process. Besides HSF1 has been reported suppressing NF-κB induced by TNF-α37
or heat shock38, the binding sites of NF-κB and HSF1 were overlapped in HIV 5′-LTR as show in the previous
report32. Therefore, phosphorylated HSF1 trimer in nucleus has possibility of occupying NF-κB binding sites,
thus the competition binding between HSF1 and NF-κB is taken place on HIV 5′-LTR. The same phenomenon
was observed in our study (Fig. 4) where salubrinal promoted much HSF1 binding on HIV 5′-LTR while little
NF-κB. Because NF-κB signaling pathway activation accelerates release of immune factors which may facilitate
T cell activation and proliferation39, the competition of NF-κB and HSF1 for binding HIV 5′-LTR may be one of
the reason accounting for latent HIV reactivation by stress without global T cell activation. It needs further study
to reveal underlying mechanism.
In conclusion, the results of the present study showed that HSF1 is an essential cellular factor for regulation
of transcription and translation in CD4+ T cells, and HIV hijacks this protein to promote viral proliferation. In
uninfected cells, HSF1 is conditionally active to facilitate protein refolding and cell survival as a primary
molecule regulating the cell stress response40. In contrast, HSF1 is constitutively active in latent T cells to participate
in latent HIV reactivation. Thus, the breadth of HSF1 biology is far greater than previously appreciated (Fig. 7).
The results in present study indicated that latent HIV reactivation mediated by HSF1 may results from many
factors such as fever, starvation or special drug stimulation. This may also account for latent HIV stochastically and
spontaneously reactivates in vivo. Bypass, HSF1 and the associated post-translational modification enzymes
identified in the present study provide further information for therapeutic development. Combined with anti-tumor
therapy, the promotion of HSF1 activation might be an appropriate solution to antagonize provirus, resulting in
the development of a cure for AIDS.
Reagents, plasmids and cell culture. EIF2α, p-eIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1
(Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling
Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE
conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C,
STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132,
BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol,
parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt,
Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat
Primary CD4+ T cell isolation and treatment. PBMCs were isolated by density gradient
centrifugation using Histopaque-1077 (Sigma-Aldrich), the peripheral blood samples from HIV positive patients and
health person were collected in The Eighth People’s Hospital of Guangzhou (Guangzhou, China) and Nanfang
Hospital (Guangzhou, China) with informed consents. The experiment was approved by the Ethical Committee
of Nanfang Hospital and performed in accordance with relevant guidelines and regulations. The HIV-infected
patients were selected based on sustained plasma viral load suppression (plasma viral loads were <20 copies/ml
for >12 months and CD4 count >350 cells/μl), and their essential information were listed in Table S3. The
primary CD4+ T cells were isolated by EasySep kit (STEMCELL Technologies Inc. Vancouver, BC, Canada)
according to manufacturer’s instruction from PBMCs and the purity was >90%. 4 × 106 cells were incubated with 200
μΜ salubrinal or 200 ng/ml plus 2 μM ionomycin for 48 h. Then cells were collected to be conducted total RNA
extraction and real time-PCR experiment with HIV 5′-LTR primers as follow.
Cell viability assay. Cell viability was evaluated by CCK-8 kit (Dojindo Molecular Technologies, Inc.,
Japan). J-Lat 10.6 and PBMCs plated as 1 × 106 cells and 2 × 106 per well respectively in 96-well plate were
incubated with compounds for 48 h. Then 10μl of CCK-8 reagent were added to 100 μl of cell culture mixture and
incubated for additional 4 h at 37 °C. Optical density (OD) was recorded at wavelength of 450 nm on a micro-plate
reader (TECAN, Swiss).
Flow cytometry (FCM). After drug treatment, the cells were analyzed by flow cytometry to calculate the
percentage of GFP-positive cells using the FITC channel (BD FACS Canto II, San Jose, CA) to determine the level
of HIV-1 gene expression. Cell surface staining was performed at 4 °C for 30 min in the dark using CD25-PE- and
CD69-APC-conjugated antibodies after drug treatment for 24 h. The cells were washed twice with phosphate
buffered saline (PBS) and analyzed through dual channel with fluorescence compensation. The fluorescence values
and dot plots were analyzed using FlowJo 7.6 software (Treestar, San Carlos, CA).
Real-time PCR. J-Lat 10.6, U1, ACH2 and primary CD4+ T cells were treated with salubrinal (200 μM) for
different times, and total RNA was extracted using TRIZOL (Invitrogen, Carlsbad, CA). Reverse transcription
was performed using the PrimeScript RT reagent Kit with gDNA Eraser (TAKARA, Japan). Real-time PCR was
performed using SYBR Select Master Mix (TAKARA) on the 7500 Real-Time PCR System (Applied Biosystems,
Foster City, CA) using a standard two-step procedure (denature: 95 °C/15 sec, anneal/extend: 60 °C/1 min, 40
cycles). The primers are list in supplemental Table S1. The 2−ΔΔCT method was adopted to analyze relative gene
expression levels and GAPDH gene was made as reference gene in all experiments.
Protein extract and Western blot analysis. After treatment, the cells were lysed in RIPA (50 mM
Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton-X-100, 0.25% sodium deoxycholate, 0.1%
SDS) containing a 1 × protease and phosphatase inhibitor cocktail (Merck Calbiochem) and incubated on ice for
10 min, followed by centrifugation at 12,000g for 10 min at 4 °C. The supernatant was collected as a whole protein
extract. The nucleoprotein was extracted using NE-PER nuclear and cytoplasmic extraction reagents (Thermo
Fisher Scientific, Carlsbad, CA) according to the manufacturer’s protocol. Total protein was quantified, and a
standard curve was generated. SDS sample buffer was added to each sample, followed by denaturation at 100 °C
for 10 min, electrophoresis for 1.5 h on a 10% polyacrylamide gel to separate the proteins, transfer onto PVDF
membranes, and co-incubation with primary and secondary antibodies conjugated to HRP. Subsequently, ECL
substrate was added, and the blot was exposed to film to develop the image.
Chromatin Immunoprecipitation (ChIP). J-Lat 10.6 cells (6 × 106 ) were treated with salubrinal or
co-treated with KRIBB11 for 2 hours, and TNF-α was made as control. Cells were harvested and fixed with 0.75%
formaldehyde for 10 min at room temperature, then lysed to obtain nuclear extract and digested to obtain
chromatin segments (200–1000 bp) using the ChIP assay kit (Thermo Fisher Scientific) according to manufacturer’s
instructions. The input controls were made as 10% nuclear extract. HSF1 and RelA monoclonal antibodies (ChIP
grade) were adopted to incubate with nuclear extract respectively at 4 °C overnight, and IgG was made as negative
control. Protein A/G was added to collect the immune complex and incubate with shaking for 1 h at 4 °C. DNA
were purified and recovered for RT-PCR as the kit instructions. The sequence of primers used for PCR
amplification of HIV-LTR is as following: forward (−183) 5′-GCCTCCTAGCATTTCGTCACAT-3′ and reverse (−18)
5′- GCTGCTTATATGTAGCATCTGAGG-3′. The amplified DNA products (166 bp), which contained the
cis-acting element for both HSF1 and NF-κB, were subjected to 2% agarose gel electrophoresis and images were
captured under the UV transilluminator (Alpha Innotech, CA, USA).
Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP). Approximately 5 × 106 J-Lat 10.6
cells were treated with salubrinal or co-treated with KRIBB11 or C646 for 2 h, and protein extracts were obtained
after the addition of 300 μl of IP lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100) and lysis on ice
for 30 min. The samples were centrifuged at 14,000rpm for 10 min, and 1 μg of mouse or rabbit IgG was added to
the supernatant followed by incubation for 30 min at 4 °C. Subsequently, 30 μl of protein A/G plus-agarose (Santa
Cruz) was added, and the mixture was incubated for another 30 min at 4 °C during the precleaning step. After
centrifugation at 2500 rpm for 5 min to obtain supernatant, 2 μg of antibody (HSF1 or acetyl-lysine) was added
to 300 μg of protein and incubated with shaking at 4 °C overnight. The input sample contained 20μg of protein.
The next day, 30μl of protein A/G plus agarose was added, and the mixture was incubated for another 4 h at 4 °C.
The mixture was centrifuged, washed three times with 500μl IP lysis buffer containing 0.1% PMSF, 1X loading
buffer was added to resuspend the precipitation, and the sample was then boiled at 100 °C for 10 min. Finally, the
samples were analyzed by Western blot.
HSF1 TALENs knockout. TALENs arms were designed as L2*R3 combination targets on the HSF1
gene (NCBI gene ID: 3297), and the plasmids for the TALENs left and right arms were constructed using the
FAST TALEN Kit (SIDANSAI, China). After sequencing, five plasmids were loaded into 293T cells in a L2*R3
cross combination. One pair of arms was selected as the most effective knockout group after 3 days of
puromycin screening and subsequent genomic PCR sequencing. Another round of screening was performed after
a two-week monoclonal culture. The stable knockout cell line was acquired and identified by sequencing and
Plasmid transfection and luciferase activity assays. 293T cells in the exponential growth period were
split at 5 × 105 cells per well and seeded into a 6-well plate. PNL4-3-luc, pEZ-HSF1 and its associated control
plasmid (pEZ-NG) were mixed with PEI (polyethylenimine), followed by co-incubation with the cells for 24 h. The
cells were lysed, and the relative luciferase activity was detected using a Dual-Luciferase Reporter Assay System
(Promega) according to instruction. The substrate and luminescence were scanned using a microplate reader with
a white plate. Relative transcription activity was normalized according to renilla luciferase activity then compared
with pNL4-3-luc and pEZ-NG co-transfection group.
Statistical analysis. All the experiments described were independently repeated at least three times to
verify reproducibility. The data were analyzed using Prism 5.0 software, and the error bars are presented as the
means ± SD from at least three independent experiments. The statistical analysis of the experimental data was
performed using one-way ANOVA with Dunnett’s test, and the P value defined as *p < 0.05, **p < 0.01, and
***p < 0.001.
The authors would like to thank Dr. Shibo Jiang and Huanzhang Zhu at Fudan University, China, for kindly
gifting many essential cells and reagents. This work was financially supported by grants from the Natural Science
Foundation of China (31370781) to S.L., the National S&T Key Special Foundation (2014ZX09509001-004) to
S.L., and the China Postdoctoral Foundation (Grant No. C1091013) to W.Z.
W.Z. and X.-Y.P. planned and performed the experiments; X.-Y.P. drafted the manuscript; X.-Y.Z., J.L., M.-M.L.
and X.-T.S. analyzed the data; S.W.L. designed the study, supervised the progress and revised the manuscript.
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Pan, X.-Y. et al. Heat Shock Factor 1 Mediates Latent HIV Reactivation. Sci. Rep. 6,
26294; doi: 10.1038/srep26294 (2016).
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