Expression of Human Endogenous Retrovirus Type K (HML-2) Is
Activated by the Tat Protein of HIV-1
Marta J. Gonzalez-Hernandez,a,bMichael D. Swanson,a,b* Rafael Contreras-Galindo,aSarah Cookinham,aSteven R. King,c
Richard J. Noel, Jr.,fMark H. Kaplan,aand David M. Markovitza,b,d,e
Department of Internal Medicine,aDepartment of Microbiology and Immunology,cand Programs in Immunology,bCancer Biology,dand Cellular and Molecular Biology,e
University of Michigan, Ann Arbor, Michigan, USA, and AIDS Research Program and Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Ricof
of years (62, 117), HERVs now exist in the genome in proviral
forms (131) consisting of the basic retroviral genes (gag, pro, pol,
and env) flanked by two long terminal repeats (LTRs) (8) formed
during reverse transcription, with the 5= LTR serving as the viral
transcriptional promoter. Most of these proviral sequences have
been rendered nonfunctional through the passage of time due to
the acquisition of multiple inactivating mutations and deletions
ing to their expression in human cells (41, 62). The endogenous
retrovirus HERV-K (HML-2), for example, has been shown to be
transcriptionally active (10, 62, 63, 103, 111, 117, 139). It is the
most recent entrant into the human genome (9, 128), having last
years ago (8), and it is the only subfamily of endogenous retrovi-
ruses with conserved, and therefore potentially functional, open
reading frames (ORFs) for all viral proteins (8, 83). There are
approximately 91 full-length copies of HERV-K (HML-2) (117)
per haploid genome and thousands of solitary LTRs. Copies of
(8, 13). HERV-K (HML-2) also encodes the accessory oncogenes
np9 and rec (nuclear protein of 9 kDa and regulator of expression
encoded by cORF) (4, 13, 14, 78), which are expressed, respec-
tively, by the two types of HERV-K (HML-2), type 1 and type 2.
of the envelope gene in type 1 (8, 117).
Endogenous retroviral elements can be involved in physiolog-
ical processes, such as those regulating the transcription of genes
such as INSL4, ?1,3-GT, endothelin B receptor, and tissue-spe-
cific salivary amylase (11, 35, 62, 84, 124). In addition, expression
of certain HERV proteins has important physiological functions,
such as in placental development (2, 34, 44, 80), and may also
ements that make up 8% of the total human cellular DNA
tion (50, 62). However, in general, how or why HERV genes are
expressed, and the mechanisms responsible for expression, is not
clearly understood. It is known that exogenous viral infections,
viral transactivators, processes such as inflammation, chemical
to the activation and transcription of transposable genetic ele-
HERV-K (HML-2) in pathogenesis has been considered in disor-
ders such as systemic lupus erythematosus, rheumatoid arthritis,
and neuroinflammation (3, 36, 43, 53, 77, 90, 112, 113). Certain
malignancies, most commonly germ cell tumors, melanoma,
breast tumors, and prostate cancer, also show high levels of
HERV-K (HML-2) antigen expression (18, 59, 62, 104, 110, 132),
sometimes accompanied by the production of viral particles (12,
89), and yet the actual contribution of HERVs to disease remains
to be characterized.
The HERV-K (HML-2) proteins Rec and Np9 provide a po-
31, 41, 52, 67, 101). Both proteins have been shown to stimulate
c-Myc expression by binding and inhibiting the c-myc gene re-
pressor promyelocytic leukemia zinc-finger protein (PLZF ),
Received 23 December 2011 Accepted 6 May 2012
Published ahead of print 16 May 2012
Address correspondence to David M. Markovitz, firstname.lastname@example.org.
*Present address: Michael D. Swanson, University of North Carolina at Chapel Hill
School of Medicine, Chapel Hill, North Carolina, USA.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org Journal of Virologyp. 7790–7805 August 2012 Volume 86 Number 15
zinc-finger protein, another transcriptional repressor (67). In ad-
dition, Rec overexpression leads to testicular carcinoma in situ in
transgenic mice (48, 106, 107). Np9 transcripts are detected with
exists that links it to oncogenesis, Np9 has been shown to interact
with a member of the cancer-associated Notch signaling pathway
Rec and Np9 has the potential to contribute to oncogenesis.
Antibodies against HERV-K have been found in the blood of
patients with a number of different clinical conditions (8, 18, 32,
54, 72, 77). One of the highest percentages of antibodies against
these retroviruses is seen in HIV-1-infected patients, where ca.
77, 116). We and others have demonstrated that HERV-K
(HML-2) RNA levels are significantly increased in the plasma of
HIV-1-infected patients (ca. 107to 108copies/ml) compared to
healthy HIV-1-negative controls (0 to 102copies/ml) (23–25, 27,
50, 130), and we have detected HERV-K (HML-2) proteins and
viral particles in the blood of human patients with HIV-1-associ-
ated lymphoma (24, 27). HIV-1 infection of peripheral blood
mononuclear cells (PBMCs) isolated from healthy donor blood
protein (26). In addition, it has been observed that HERV-K ele-
ments are overexpressed in brain tissue of AIDS patients who de-
velop neurological complications due to increased immune acti-
vation (63). How HERV-K might be activated by HIV has
remained an open question. It is possible that the increased ex-
pression of HERV-K in HIV-1 infection is due to immunosup-
pression, but it could also be a consequence of direct interaction
with infectious HIV-1 particles or viral proteins (130). Interest-
ingly, work from the Cullen and Löwer laboratories has provided
evidence that HIV-1 Rev recognizes the cis-acting Rec response
response element of HIV-1, and can actively export HERV-K
(HML-2) RNA from the nucleus to the cytoplasm (79, 135).
The HIV-1 regulatory protein Tat is a potent transactivator of
the HIV promoter and is essential for viral replication (42). Tat is
produced in the early phase of HIV infection as a 72- or 101-
amino-acid protein; depending on the viral isolate, a truncated
86-amino-acid form can also be produced (19, 38, 42, 61, 64). In
addition to activating HIV transcription in the cell where it is
surroundings, where it can be taken up by neighboring cells and
exert effects on gene expression (38, 42, 60). In addition, HIV-1
Tat has been known to act not only on the HIV-1 and HIV-2
108, 120, 137). Interestingly, the HIV-1 Tat protein has been
effect mediated by interactions of the transcription factor TFIIIC
with the Alu promoter (60).
In view of the above, we hypothesized that the HIV-1 Tat pro-
tein provides a functional link between HIV-1 infection and the
induction of HERV-K (HML-2) gene expression by causing acti-
vation of HERV-K LTR-directed transcription. In the studies de-
cell lines and primary lymphocytes. We further demonstrate that
the HERV-K (HML-2) transcriptional promoter is responsive to
Tat and that this effect is mediated by NF-?B and NF-AT. This
may explain, at least in part, why HIV-1 infection is associated
MATERIALS AND METHODS
Plasmid constructs. The HIV-1 molecular clone pNL4-3 has been previ-
ously described (Malcolm Martin, NIH AIDS Research and Reference
Reagent Program ). The HIV-1 Tat coding plasmids pcDNA3.1-Tat72
and pcDNA3.1-Tat86 were made from the parent vector pcDNA3.1?/
Tat101-flag (PEV280; Eric Verdin, NIH AIDS Research and Reference
and Tat T23N were made through site-directed mutagenesis of the
control was made by releasing the Tat insert with BamHI, followed by
religation of the backbone. The HIV-1 Rev coding plasmid was made by
releasing the rev insert from the pRev-1 plasmid construct (Marie-Louise
Hammarskjöld and David Rekosh, NIH AIDS Research and Reference
Reagent Program ) with BamHI, and ligation into pcDNA3.1(?).
The HIV-1 Nef protein is also expressed from pcDNA3.1(?), containing
the coding region of HIV-1SF2Nef, and has been previously described (J.
Victor Garcia and John Foster, NIH AIDS Research and Reference Re-
agent Program ). The HIV-1 Vif and Vpu proteins were expressed
from pcDNA3.1(?), with both being codon optimized for expression in
(Stephan Bour and Klaus Strebel, NIH AIDS Research and Reference Re-
agent Program ). Full-length Vpr was cloned into the peGFP-C3 ex-
protein (GFP; Warner Greene, NIH AIDS Research and Reference Re-
agent Program ). A vector that expresses HIV-1 Gag derived from
Ono at the University of Michigan and has been previously described
(pCMVNLGagPolRRE ). pCMV (pCMV-PL; Bryan Cullen) was ob-
tained from Addgene (Cambridge, MA). Full-length Env from HIV-
1HXB2was cloned into pSV7D, termed pHXB2Env for simplicity, and has
been described previously (Kathleen Page and Dan Littman, NIH AIDS
Research and Reference Reagent Program ). The molecular clone
molecular clone pMtat(?) contains a termination codon (TGA) in place
and Reference Reagent Program ). The HERV-K (HML-2) LTR re-
porter construct (HERV-K LTR-luc) contains a partial version of the
HERV-K (HML-2) LTR (GenBank accession no. AF394944) cloned in
and was kindly provided by Kyung Lib Jang (Pusan National University,
Pusan, South Korea). The Renilla luciferase plasmid, pRL-CMV, was ob-
tained from Promega (Madison, WI). The luciferase construct “HERV-K
LTR,” used in mutational analyses, was made by addition through PCR
amplification of the consensus sequence TGTGGGGAAAAGCAAGAGA
to the 5= end of the partial promoter sequence of the HERV-K (HML-2)
LTR from pT81 and subcloning it into the XhoI and HindIII sites of
pGL4.10[luc2] (Promega, Madison, WI), which does not contain a tk
minimal promoter. Site-directed mutagenesis was performed on the LTR
region using the QuikChange Lightning site-directed mutagenesis kit
Y ? pyrimidine, and N ? any nucleotide) as GCTCTAYYCC. NF-AT
consensus sequence (A/T)GGAAA(A/N)(A/T/C)N to CTCTA. In cases
where NF-AT sites were embedded in NF-?B sequences, mutation to
CTCTA was used to eliminate both sites simultaneously. For analyses
involving the potential Sp1 binding site, two binding site mutants were
generated and tested, taking the consensus sequence GGGCGG(G/A)(G/
A)(C/T) and changing it to either GAGATCTGC or TTGAGGTGC. PCR
Primer Design Program (Stratagene). Sequences of the plasmids were con-
Cell culture and transient transfections. Except for 293FT (a fast-
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15 jvi.asm.org 7791
Research and Reference Reagent Program ), and Jurkat-Tat T cells
(stably expressing HIV-1 Tat; Antonella Caputo, William Haseltine, and
Joseph Sodroski, NIH AIDS Research and Reference Reagent Program
), all cell lines used were obtained from the American Type Culture
Collection (ATCC; Manassas, VA). All media were obtained from Gibco
(Invitrogen). 293FT were maintained in Dulbecco modified Eagle me-
dium, supplemented with 10% fetal bovine serum (FBS; Gibco/Invitro-
gen, Carlsbad, CA) and 100 U of penicillin and streptomycin (Gibco/
H9 (a derivative of HUT-78), and NCCIT cells were maintained in com-
cells were maintained in complete RPMI 1640 supplemented with 10%
FBS, 800 ?g of G418 (Invitrogen)/ml, and 100 U of penicillin and strep-
tomycin/ml. When needed, the cells were cultured under stimulatory
conditions with PMA (phorbol 12-myristate 13-acetate) and ionomycin,
with DMSO (dimethyl sulfoxide) as their vehicle buffer (all from Sigma-
Aldrich, St. Louis, MO). Cells with ?90% viability were transfected with
endotoxin-free plasmids [2 to 5 ?g of HIV-1-Tat, Vif, Nef, Rev, Vp(h)u,
Vpr, and GagPol plasmids, 2 to 5 ?g of empty/control vectors, or 5 ?g of
pHXB2 or pMtat(?) proviral molecular clones] using the transfection
reagents Lipofectamine 2000 (Invitrogen), SuperFect (Qiagen, Valencia,
CA), or FuGENE HD (Roche, Indianapolis, IN) according to the manu-
facturers’ protocols. Control experiments included mock transfections.
HIV-1 production and infection. Infectious HIV-1 was produced by
calcium phosphate-mediated transfection of 293FT cells (136) using
pNL4-3 (1). Tissue culture medium was harvested at 24, 36, or 48 h post-
transfection, pooled, and filtered (0.2-?m pore size) to remove cells and
stocks were determined from the reverse transcription (RT) activity (7)
(7). HIV infection of cells (2 ? 106) was conducted using 2 ml of virus in
ature. Infected cell cultures were diluted in T-25 flasks and maintained at
between 0.5 ? 106and 1 ? 106cells per ml for 7 days; then, fresh unin-
fected cells were added, and the cells were harvested after an additional
amplification was performed to confirm the infection status with the fol-
lowing primer sequences, in a one-step reverse transcription-PCR (RT-
PCR): HIV-1 Env F 1493-1516 (5=-AGGCAAAGAGAAGAGTGGTGCA
GA-3=) and HIV-1 Env R 1643-1666 (5=-CCCTCAGCAAATTGTTCTG
Luciferase assays. All transfections included Renilla luciferase as an
internal control (100 ng of pRL-CMV) to assess for variation in transfec-
tion efficiency. Transfected cells were assayed for luciferase activity using
a dual-luciferase assay kit (Promega, Madison, WI), 4, 6, 8, 12, 24, and 48
h after transfection in a Tecan GENios luminometer plate reader (Phenix
luciferase signal and are expressed as standardized luciferase units. The
amounts of plasmids used for transfections were as follows: 2.5 ?g of
2 to 5 ?g of Tat expression plasmid or 2 to 5 ?g of empty/control vector.
Control experiments included mock transfections (Lipofectamine 2000
reagent alone) and no transfection (for background subtraction).
Isolation and culture of primary cells. PBMCs were obtained by ve-
nipuncture from healthy donors and monocytes were separated from pe-
ripheral blood lymphocytes (PBLs) by differential adhesion to plates as
previously described (119). PBLs were washed three times with phos-
tinin (PHA-P; Sigma-Aldrich)/ml for 3 days in RPMI 1640 complete me-
dium containing 10% heat-inactivated FBS and 10 U of interleukin-2
Addition of exogenous Tat protein. The purified recombinant 86-
amino-acid form of the HIV-1 Tat protein was obtained from the NIH
AIDS Research and Reference Reagent Program (the late John Brady and
DAIDS, NIAID, ) or from ProSpec Protein Specialists (catalog no.
HIV-129; ProSpec-Tany TechnoGene, Ltd., East Brunswick, NJ). The
protein was resuspended in sterile PBS (Gibco/Invitrogen) containing 1
(both from Sigma-Aldrich), de-aerated, and protected from light. Tat
protein was added in a range of concentrations to cells for the specified
time points, as indicated in the text and figure legends.
RNA extraction and real-time RT-PCR. Total cellular RNA was iso-
lated from cells using the RNeasy minikit (Qiagen) and subjected to
RNase-free DNase treatment (Qiagen) for 15 min at room temperature.
The RNA concentration and purity were measured using a spectropho-
tometer, calculating the 260/280 ratio. RNA integrity (as well as the ab-
sence of DNA contamination) was confirmed by one-step RT-PCR using
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) amplification
with primers that can bind both genomic and cDNA, under the PCR
conditions described below, as well as “no RT” controls. If DNA contam-
ination was detected, another round of DNase treatment was performed
manufacturer’s protocol. To assess the difference in HERV-K (HML-2)
RNA transcript expression levels, we performed quantitative real-time
RT-PCR using a QuantiTect SYBR green RT-PCR kit (Qiagen) or a Bio-
Rad iScript one-step RT-PCR kit with SYBR green (Bio-Rad, Hercules,
CA). Briefly, 100 to 500 ng of total cellular RNA and 0.5 ?M concentra-
tions (each) of HERV-K (HML-2) gag primers (forward primer, 5=-AGC
AGGTCAGGTGCCTGTAACATT-3=; reverse primer, 5=-TGGTGCCGT
AGGATTAAGTCTCCT-3=), HERV-K (HML-2) rec primers (forward
GGTACACCTGCAGACACCATTGAT-3=), or HERV-K (HML-2) np9
primers (forward primer, 5=-AGATGTCTGCAGGTGTACCCA-3=; re-
verse primer, 5=-CTCTTGCTTTTCCCCACATTTC-3=) were used in a
20-?l reaction. RNA was reverse transcribed for 30 min at 50°C. PCR
40 cycles with optimal conditions as follows: 94°C for 15 s, 60°C for 30 s,
and 72°C for 10 s, as well as optimized data collection steps at 81°C for 10
generated by primer dimers by a melting-curve analysis. The data were
fication was used to normalize samples to an endogenous reference gene,
as stated in the figure legends.
Western blot and protein band analysis. The antibodies used in the
present study, and their respective dilutions, are as follows: mouse anti-
HERV-K Gag (HERM-1841-5, 1:1,000 dilution) and mouse anti-
HERV-K capsid (HERM-1831-5, 1:200 dilution), both from Austral Bio-
1:25,000 dilution; Abcam, Cambridge, MA); and mouse anti-HIV-1 Tat
(1D9; NIH AIDS Research and Reference Reagent Program, Bethesda,
MD). After transfection, or recombinant protein addition, Jurkat T cells
were washed twice with PBS and then lysed with hot 2% sodium dodecyl
sulfate (SDS) buffer (Fisher Scientific, Pittsburgh, PA). The cell lysates
were boiled and centrifuged to eliminate DNA-associated viscosity, and
reagent kit (Pierce/Thermo Scientific, Rockford, IL). Equal protein con-
centrations were loaded and separated on SDS–15% polyacrylamide gels
and blotted onto polyvinylidene difluoride membranes. The membranes
Fischer Scientific, Pittsburgh, PA) for 2 h at room temperature. All of the
antibodies used were incubated in blocking solution with the blotted
at room temperature). The membranes were washed three times in PBST
and blocked with 5% goat serum for 30 min at room temperature. When
necessary, the bound primary antibody was incubated with an HRP-con-
jugated goat anti-mouse secondary antibody for 1 h. Signal was detected
using the Super Signal West Pico system (Pierce/Thermo Scientific).
Gonzalez-Hernandez et al.
jvi.asm.org Journal of Virology
Semiquantification of protein levels was performed by digitization of X-
ray films using the Typhoon FLA 7000 scanner (GE Healthcare Life Sci-
ences, Pittsburgh, PA) and subsequent analysis of the gray values of the
bands in the resulting images. The ImageQuant TL software was used for
analysis of the digitized Western blot images. This software allows the
measurement of band volume (average optical density of the band times
its area) of the band of interest. Background subtraction was performed
using the “image rectangle” method as a user-defined area within the
of interest. Bands were fitted as tightly as possible, and band volumes are
plotted as arbitrary units.
Identification of potential transcription factor binding sites in the
LTR of HERV-K (HML-2). Analyses of the HERV-K (HML-2) LTR for
potential transcription factor binding sites were performed using the on-
line prediction software tools ALGGEN PROMO and version 8.3 of
TRANSFAC (BioBase Co., Beverley, MA) (40, 85).
vided by Paul J. Chiao, MD Anderson Cancer Center, Houston, TX),
referred to here as I?B? DN (dominant negative), was cotransfected into
Jurkat T cells with the HERV-K (HML-2) LTR-luciferase construct with
or without Tat (using Renilla luciferase as an internal transfection con-
trol). The luciferase activity was measured 48 h after transfection. I?B?
DN encodes a phosphorylation site and a degradation site mutant I?B?
chain, inhibits translocation of NF-?B from the cytosol to the nucleus
upon activation, and has a dominant-negative effect on NF-?B function
(33, 46, 47).
NF-?B inhibition was also accomplished through a 1 h pretreatment
trihydroxycoumestan; Sigma-Aldrich) in DMSO, followed by treatment
phosphorylation and degradation of I?B? (70).
NF-AT inhibition assays. Jurkat T cells cotransfected with the
HERV-K (HML-2) LTR reporter construct and the Tat expression vector
were treated with various doses of the immunosuppressant drug cyclo-
was measured 48 h after transfection, as noted above and in the figure
Specific NF-AT inhibition was accomplished through a 1-h pretreat-
ment of experimental samples with 1 ?M 11R-VIVIT in DMSO (Calbi-
ochem, La Jolla, CA), followed by treatment with recombinant Tat pro-
tein or its buffer control. 11R-VIVIT is a cell-permeable version of the
specific NF-AT inhibitor (VIVIT) that is modified at the C terminus with
ChIP assays. To examine interactions of NF-?B and NF-AT with the
HERV-K (HML-2) LTR, we performed chromatin immunoprecipitation
bad, CA) according to the manufacturer’s protocol. Briefly, experimental
samples were lysed and subjected to enzymatic shearing of the DNA
(random cleaving). Digestion was performed for 4 h at 37°C, with
intermittent vortexing. A 2-?g portion of specific antibody for NF-?B
(H-119X; Santa Cruz Biotechnology, Santa Cruz, CA), NF-AT (7A6;
Santa Cruz Biotechnology), or vimentin (V9; Santa Cruz Biotechnol-
ogy) or IgG isotype control antibodies (rabbit IgG or mouse IgG;
Sigma-Aldrich) were used for each immunoprecipitation, with over-
night incubation. Protein G-beads were added to the overnight incu-
bation as well. Beads were washed three times in ChIP buffer, after
which elution of the digested chromatin, reversion of cross-linking,
and proteinase K treatment was performed. Immunoprecipitated
DNA was detected by PCR, using 5 ?l of eluate as a template, to verify
success of the precipitation. For amplification of the HERV-K
(HML-2) LTR transcription factor binding site areas of interest, the
following primers were used (their binding areas are depicted in
Fig. 6): KLTR ChiP primer set 1 fwd (5=-TGTGGGGAAAAGCAAGAG
A-3=), KLTR ChiP primer set 1 rev (5=-GGTCACAGAATCTCAAGGCA
G-3=), KLTR ChiP primer set 2 fwd (5=-GTGACCTTACCCCCAACCCC
3=), KLTR ChiP primer set 3 rev (5=-CGGGTATCGGGCTGGGGGACG-
3=), KLTR ChiP primer set 4 fwd (5=-CCCTGGGCAATGGAATGTCTC
G-3=), KLTR ChiP primer set 4 rev (5=-GCTGCCCGCAGGTCCCACCT
C-3=), KLTR ChiP primer set 5 fwd (5=-TGGTTCCCCGGGTCCCCTTA
T-3=), and KLTR ChiP primer set 5 rev (5=-CCTACACACCTGTGGGTG
Real-time PCR was performed with the Bio-Rad iQ SYBR green Su-
permix (Bio-Rad) with a 0.3 ?M final primer concentration in a 20-?l
final reaction volume and the following cycle conditions: 1 cycle of 95°C
30 s, followed in turn by 1 cycle of 72°C for 10 min. A melting-curve
analysis was also performed to verify specific product amplification.
as controls, with PCR conditions as stated above.
Statistical analysis. The mean number of HERV-K (HML-2) mRNA
and standardized luciferase units between Tat treatments and controls
normally distributed values. Two-tailed P values were considered signifi-
cant at P ? 0.05.
HERV-K (HML-2) RNA expression is increased in HIV-1-in-
fected cell lines. Recent studies by our group and others have
shown that HIV-1 infection increases HERV-K (HML-2) gene
expression, both in cell culture and in patients (22–27, 50). How-
ever, the underlying mechanism for this increased expression has
remained unknown. To begin to address this issue, we first ascer-
after HIV-1 infection of 2 different T cell lines. Quantification of
per 500 ng of total RNA (data not shown). These levels increased
up to ?20-fold with HIV-1 infection (Fig. 1). Higher expression
of HERV-K (HML-2) gag RNA was consistently seen in all HIV-
1-infected cells compared to their uninfected counterparts (P ?
0.001, Fig. 1). Interestingly, uninfected Jurkat T cells that stably
express the Tat protein from HIV-1 (Jurkat-Tat) showed 8-fold
more HERV-K (HML-2) gag RNA expression than did Jurkat T
that Jurkat-Tat cells have higher HERV-K (HML-2) gag RNA ex-
esized that the HIV-1 Tat protein might play a role in activating
HERV-K (HML-2) expression during HIV-1 infection.
HIV-1 Tat and Vif independently cause an increase in
HERV-K (HML-2) gag RNA expression. To ascertain whether
our hypothesis that HIV-1 Tat activates the synthesis of HERV-K
(HML-2) gag RNA is correct, we first transfected different cell
types with plasmids encoding each of the regulatory, accessory,
and structural proteins from HIV-1 and measured the levels of
cellular HERV-K (HML-2) gag RNA after 24 and 48 h. We used a
transfectable line (293FT), and a cell line known for its high ex-
pression of HERV-K (HML-2) transcripts and proteins (NCCIT
teratocarcinoma cells). Expression of Tat (from a plasmid encod-
ing the full-length, 101-amino-acid form) or Vif yielded a signif-
icant increase in HERV-K (HML-2) RNA expression (Fig. 2A).
The presence of Tat or Vif increased HERV-K (HML-2) gag RNA
by about 21- or 15-fold, respectively, compared to untreated Jur-
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15 jvi.asm.org 7793
kat cells (P ? 0.01). Similar responses to Tat and Vif were seen in
293FT cells, whereas the teratocarcinoma NCCIT showed a re-
sponse only to Vif (Fig. 2A). Similar increases in HERV-K
(HML-2) gag RNA expression were observed 48 h after transfec-
tion (data not shown). The expression of any of the other HIV-1
proteins resulted in no significant increase in HERV-K (HML-
To determine whether Tat and Vif synergistically increase
HERV-K (HML-2) transcript levels, we cotransfected plasmids
encoding these proteins and observed the effect on transcription
after 24 h. For both Jurkat T cells and 293FT, Tat and Vif had an
additive, not synergistic, effect with regard to HERV-K (HML-2)
transcription, whereas in NCCIT no significant difference was
seen compared to Vif-induced expression alone (Fig. 2B). Tat
functionality was verified in NCCIT cells in parallel cotransfec-
tions using an HIV-1 LTR-luciferase reporter assay (Fig. 2B, in-
set). These data show that both Tat and Vif can activate HERV-K
(HML-2) transcription and suggest that Tat alone is sufficient for
activation in HIV-1-relevant targets of infection (i.e., Jurkat T
To further test the importance of Tat in HIV-1-mediated acti-
vation of HERV-K (HML-2), we transfected an HIV-1 infectious
protein [pMtat(?)] into Jurkat T cells and 293FT cells and mea-
sured HERV-K (HML-2) gag RNA 48 h later. As can be seen in
Fig. 2C, Tat expression in Jurkat T cells is important for higher
HERV-K (HML-2) RNA expression, since its absence greatly di-
minishes levels of transcripts (by more than half, P ? 0.05). Of
proteins. However, these data are consistent with the observation
that HIV-1 Tat expression is sufficient to increase HERV-K
(HML-2) transcript expression.
Recombinant HIV-1 Tat increases HERV-K (HML-2) ex-
pression. We next analyzed whether addition of physiologically
relevant levels of recombinant HIV-1 Tat to cells could activate
HERV-K (HML-2) transcription. We took advantage of the fact
that, unlike most transcription factors, HIV-1 Tat is able to cross
intact cellular membranes when it is present in the extracellular
measured HERV-K (HML-2) gag RNA expression showed that
8 h (13-fold increase) and then gradually declined (Fig. 3A, left
panel). Recombinant Tat activity was further verified in parallel
experiments by measuring reporter activity from Jurkat T cells
transfected with a vector containing the HIV-1 LTR fused to the
luciferase reporter gene, which showed the protein to be fully ac-
HERV-K (HML-2) gag activation, we heat denatured Tat and
added it to cells (endotoxin is highly heat stable, whereas Tat is
not). No significant increase in HERV-K (HML-2) gag RNA was
detected under these conditions, suggesting that activation of
and not to endotoxin (Fig. 3A).
cell type alone, since we also detected significantly increased
HERV-K (HML-2) transcripts for gag, rec, and np9 in HUT-78
lymphoblasts, U-937 monocytes, and 293FT fibroblasts after Tat
was added for 8 h (Fig. 3B). This effect was not seen with all cell
types tested, since the teratocarcinoma cell line NCCIT did not
show any significant increase in HERV-K (HML-2) gene expres-
of effect was not due to the cells being nonpermissive or nonre-
sponsive to Tat comes from parallel experiments involving trans-
fection of the HIV-1 LTR-luciferase reporter vector, and subse-
quent Tat exposure, which showed that Tat could be internalized
and activates the HIV-1 LTR (Fig. 3B, inset).
Since the experiments described above were all performed in cell
most relevant to HIV infection. PBLs from healthy individuals were
from unstimulated PBLs showed that Tat treatment leads to expres-
to rise and peaks by 12 h, with an ?10-fold increase over cells not
PHA and IL-2 alone for 3 days activates transcription of HERV-K
(HML-2) (data not shown), but this effect was sustained for only
about a total of 74 h in culture (3 days of prestimulation and then
Total cellular RNA was isolated from cells that were infected with HIV-1NL4-3
(for 1 week) or left uninfected. RNA was amplified using primers specific for
HERV-K (HML-2) gag through one-step qRT-PCR and quantified using a
standard curve generated by amplification of HERV-K (HML-2) gag RNA
standards. The data are expressed as the fold increase over uninfected cells,
with uninfected cells shown as normalized to 1 for simplicity of comparison.
The rightmost panel shows Jurkat-Tat HERV-K (HML-2) gag RNA levels
levels normalized to 1. HIV-1 env and GAPDH one- step RT-PCR amplifica-
the material (NTC, nontemplate control). Error bars indicate the standard
was calculated by comparing infected samples with their uninfected counter-
parts using a Student t test and significant results are indicated (*, P ? 0.005).
Gonzalez-Hernandez et al.
jvi.asm.orgJournal of Virology
experimental treatments), with RNA expression returning to basal
Since our data show that Tat activated HERV-K (HML-2)
transcription, we next assessed whether the increases in HERV-K
Using untreated Jurkat T cell lysate with a mix of commercially
available antibodies against the full-length HERV-K (HML-2)
Gag and its capsid form, little protein expression was detected in
FIG 2 HIV-1 Tat and Vif proteins activate HERV-K (HML-2) RNA expression. (A) Total cellular RNA was isolated from cells 24 h after transfection with plasmids
expression construct. Full-length Vpr was cloned into the peGFP-C3 expression vector and is fused to GFP. Full-length HIV-1 Gag and Pol were expressed from the
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15jvi.asm.org 7795
FIG 3 Recombinant HIV-1 Tat activates HERV-K (HML-2) gene expression. Total cellular RNA was isolated from cells that were subjected to different Tat
left panel shows the HERV-K (HML-2) gag RNA expression from Jurkat T cells treated with 100 ng of purified Tat protein/ml for the specified periods of time.
The right panel shows the results for Jurkat T cells transfected with an HIV-1 LTR-luciferase reporter construct, which 24 h later were treated with 100 ng of
luciferase and are expressed as standardized luciferase units. Denatured Tat protein and the Tat vehicle buffer were used as negative controls, and only the 24-h
time point is shown for those as representative results. (B) RNA expression of the HERV-K (HML-2) genes rec, np9, gag, envelope type 1, and envelope type 2 in
different cell lines after 8 h of treatment with 100 ng of Tat protein/ml. The inset shows the Tat activity verified by transfection with an HIV-1 LTR-luciferase
Gonzalez-Hernandez et al.
jvi.asm.org Journal of Virology
untreated cells, as has previously been reported (16). When Tat
was transfected into Jurkat T cells, full-length HERV-K (HML-2)
Gag protein was still minimally expressed but the cleaved capsid
sion over the empty vector control (Fig. 3D and E). A similar
added to the cells, although treatment with the vehicle buffer
(which contains the reducing agent DTT) in this case also in-
creases the expression to some degree (Fig. 3D). Protein increases
seen with Tat, these results show that Tat can substantially in-
crease the expression of HERV-K (HML-2) Gag, and this protein
is detected in its capsid form after it is further processed by a
well as the cleaved capsid (12, 16).
Taken together, these observations demonstrate that HIV-1
and with significant transcriptional activation seen in primary
lymphocytes, a major target for HIV infection.
Since HIV-1 Tat is known to act upon both viral and cellular
promoters to increase or decrease gene expression (17, 19, 60, 73,
74, 102, 108, 115, 137), we tested whether the effect of Tat on
HERV-K (HML-2) occurred at the level of the transcriptional
promoter driving the expression of the luciferase reporter gene,
Jurkat T cells were cotransfected with plasmids encoding one of
three isoforms of Tat (72, 86, or 101 amino acids). This was done
since it is known that all isoforms do not necessarily behave the
same with regard to cellular gene activation (e.g., full-length Tat
101, but not one-exon Tat 72, represses MHC-I expression) (19,
56, 133). The expression of Tat RNA and protein was confirmed
by RT-PCR and immunoblot analyses (data not shown), and its
functional integrity was confirmed by showing transactivation of
the HIV-1 LTR in luciferase reporter gene assays as described
above. Consistent with the findings shown in Fig. 2 and 3 above,
we found that all HIV-1 Tat isoforms transactivated the HERV-K
(HML-2) promoter with almost equal efficiency, approximately
(Tat 86 isoform) to Jurkat T cells that were transfected with the
HERV-K (HML-2) LTR reporter construct, which led to an in-
crease in luciferase activity of approximately 4- to 8-fold over the
signal generated by the buffer control alone (Fig. 4B). Tat showed
little effect on the HERV-K (HML-2) promoter after transfection
into 293FT or NCCIT cells (data not shown), which suggests that
the Tat-mediated stimulation of HERV-K (HML-2) is cell type
specific. Interestingly, the degree of activation seen from the
was consistent with the observed Tat-induced increases in gag
RNA. These promoter activation data thus support our observa-
In order to understand whether HERV-K (HML-2) promoter
activate transcription from the HIV-1 LTR, we cotransfected the
HERV-K (HML-2) LTR-luciferase constructs with two different
C22G contains a mutation in a cysteine at position 22 (to a gly-
cine) in the transactivation domain, which renders it unable to
interact with cyclin T1, and thus it is HIV-1 LTR activation defi-
cient (49, 88). Tat T23N, on the other hand, contains a mutation
in the threonine at residue 23 (to asparagine) that increases Tat’s
ability to activate the HIV-1 LTR by increasing binding of Tat to
the cellular kinase-positive transcription elongation factor b (P-
drive transcription from the HERV-K (HML-2) promoter,
These data show that activation of HERV-K (HML-2) transcrip-
tion by Tat occurs in a different manner than that of HIV-1, and
does not appear to involve Tat’s interaction with Cyclin T1 or
P-TEFb. This is consistent with the data described below.
Activation of the HERV-K (HML-2) promoter by Tat is me-
diated by NF-?B and NF-AT. In addition to stimulating HIV
transcriptional elongation through its interactions with cyclin T1
and P-TEFb, Tat is known to activate cellular genes through reg-
ulation and/or interaction with upstream cellular transcription
factors. Furthermore, it has been shown that Tat can, in the ab-
sence of a functional TAR, remain an important factor for HIV-1
transcription via Sp1 sequence elements in the U3 promoter re-
gion (29). Tat can additionally interact directly with NF-?B, with
this interaction not only demonstrating TAR-independent trans-
activation in HIV-1 but also pointing toward a mechanism of
Tat-mediated modulation of cellular genes (28). To understand
the mechanism by which Tat activates HERV-K (HML-2) LTR-
directed transcription, we analyzed the sequence of the promoter
for potential transcription factor binding sites. Utilizing the
algorithm TRANSFAC database software (BioBase, Beverley,
MA), we analyzed the promoter sequence and found that a num-
ber of transcription factors might potentially interact with the
HERV-K (HML-2) promoter, including AP-1, CREB, CEBP (C/
luciferase units. (C) HERV-K (HML-2) gag RNA expression from PBLs. PBLs were split in half and either treated with 100 ng of Tat protein/ml for 8 h or
using the 2???CTmethod, and the relative expression is plotted as the fold increase over untreated cells. Error bars indicate the SD for the results of three
independent experiments. Significance was calculated by comparing Tat treatments to buffer controls, at the same time points, using a Student t test, and
in Jurkat T cells after treatment with HIV-1 Tat. For the detection of HERV-K (HML-2) Gag protein, two monoclonal antibodies were used simultaneously:
anti-HERV-K Gag and anti-HERV-K capsid. The left panel shows the HERV-K (HML-2) Gag protein expression in cell lysates 24 h after transfection with Tat
lysate (“Unt”). Respective Gag proteins and sizes are shown. Tat protein expression in transfected cells is shown, and ?-actin protein expression is shown as a
loading control. (E) Densitometry analysis of the HERV-K (HML-2) Capsid protein bands seen in the Western blots in panel D.
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15jvi.asm.org 7797
EBP?), c-Rel, NF-AT, CEBP?,NF-?B(p50:p52), Rel-A, p53, YY1,
decided to focus on ones previously shown to be particularly as-
sociated with HIV-1 Tat activation: Sp1, NF-?B, and NF-AT (30,
51, 69, 86, 87, 118, 129, 134). Two potential NF-?B binding sites,
with NF-AT sites embedded in them (here referred to as ?B1/N1
and ?B3/N3), are found in the most upstream part of the U3
region of the promoter, along with single NF-AT (N2), NF-?B
(?B2), and Sp1 sites (Fig. 5A). The R region contains a lone NF-
?B/NF-AT site (?B4/N4), and the U5 region has two potential
NF-AT binding sites present (N5 and N6, Fig. 5A).
In view of the multiple NF-?B sites found in the HERV-K
mediated activation of the HIV-1 promoter (30, 76), we tested
whether NF-?B mediates the Tat effect on the HERV-K (HML-2)
LTR. To do so, we first examined whether a dominant-negative
inhibitor of NF-?B nuclear translocation would block Tat stimu-
lation of the HERV-K (HML-2) promoter. This dominant-nega-
tive construct (referred to in the figure as I?B? DN) codes for the
inhibitor of NF-?B alpha (I?B?) and sequesters NF-?B in the
tion. At 48 h after cotransfection, the activation of the HERV-K
(HML-2) promoter construct in response to Tat was significantly
decreased, by approximately 65%, in the presence of the NF-?B
inhibitor (Fig. 5B, left panel, P ? 0.05). This dependence on
NF-?B was corroborated in experiments in which Jurkat T cells
were pretreated for 1 h with a specific, irreversible inhibitor of
IKK? and ? kinase activity, wedelolactone (70) before the addi-
tion of recombinant Tat protein. After 6 h, RNA was isolated and
quantitated by qRT-PCR. Figure 5B (right panel) shows that in-
hibition of NF-?B diminishes the increase in HERV-K (HML-2)
gag RNA seen in response to Tat by half (P ? 0.01) but does not
abolished, in the presence of wedelolactone. Thus, NF-?B medi-
ates, in part, activation of HERV-K (HML-2) by Tat.
Since the HERV-K (HML-2) LTR also contains potential
NF-AT binding sites, NF-AT activation could additionally con-
FIG 4 HIV-1 Tat activates the HERV-K (HML-2) promoter by a different mechanism than it uses to activate HIV-1. (A) Jurkat T cells were transfected
with a HERV-K (HML-2) LTR-luciferase construct (except for the luciferase backbone construct pT81 and mock samples) and cotransfected with the
corresponds to untreated cells. Activation of the luciferase construct was measured 24 h after transfection, normalized to Renilla luciferase signal, and
shown as standardized luciferase units. The fold induction was calculated over the empty vector, pcDNA3.1. Control experiments included a mock
transfection and transfection of the backbone luciferase vector (pT81) alone or with a Tat-expressing vector. (B) Jurkat T cells were transfected with the
HERV-K (HML-2) LTR-luciferase construct, or a similar HIV-1 LTR luciferase construct as a positive control, and 24 h after transfection were treated
with 500 ng of purified Tat protein/ml for the specified periods of time. The buffer control is an 8-h treatment. (C) Jurkat T cells were transfected with the
HERV-K (HML-2) LTR- or HIV-1 LTR-luciferase reporter construct and cotransfected with the Tat mutants C22G or T23N, and the cells were harvested
at 24 h. These mutants were expressed from the pcDNA3.1 expression vector and are either unable to activate transcription from the HIV-1 LTR (Tat
C22G) or cause an increase in HIV-1 LTR transcriptional activity (Tat T23N). The data are shown as standardized luciferase units. Error bars indicate the
SD from three independent experiments. Significance was calculated using a Student t test comparing Tat transfection/treatment to pcDNA3.1 (A and C)
or buffer control (B), and significant results are indicated (*, P ? 0.05; **, P ? 0.01).
Gonzalez-Hernandez et al.
jvi.asm.orgJournal of Virology
panel shows the results for a dominant-negative construct coding for the inhibitor of NF-?B alpha (I?B-? DN) that was cotransfected into Jurkat T cells with the
luciferase, and expressed as standardized luciferase units. For the right panel, Jurkat T cells were pretreated for 1 h with 10 ?M wedelolactone (a specific inhibitor of
and Ionomycin treatments served as positive controls for transcription factor activation. Error bars indicate the SD from three independent experiments. Significance was
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15jvi.asm.org 7799
tribute to HERV-K (HML-2) Tat-driven expression and might
compensate for the absence of NF-?B activity. We therefore ex-
amined whether inhibiting NF-AT activation would also result in
diminished HERV-K (HML-2) responsiveness to Tat. Treatment
of Jurkat T cells that were cotransfected with the HERV-K
(HML-2) reporter construct and Tat with the immunosuppres-
sive drug cyclosporine (a calcineurin inhibitor that prevents de-
phosphorylation of NF-AT and therefore its activation) showed a
ose-dependent inhibition of Tat-mediated HERV-K (HML-2)
promoter activation (Fig. 5C, CsA, left panel). This suggests that
mide] assay was performed on cells treated with the described
doses of cyclosporine and showed no significant cell death at any
that cyclosporine is not an NF-AT-specific drug, we also pre-
treated Jurkat T cells with 11R-VIVIT, a cell-permeable peptide
inhibitor specific for NF-AT (92) for 1 h, added recombinant Tat
protein for 6 h, and then isolated RNA. We observed a reduction
in gag RNA to about half of the starting levels in cells treated with
the NF-AT inhibitor in the presence of Tat (Fig. 5C, right panel).
Treatment with both wedelolactone and 11R-VIVIT simultane-
gag RNA levels, since this treatment decreased expression by ap-
proximately 75% (Fig. 5D). Overall, these data show that both
scription from the HERV-K (HML-2) promoter.
To verify the physical interaction of NF-?B and NF-AT with
the HERV-K (HML-2) promoter, we performed ChIP assays. Af-
ter Jurkat T cells were treated with HIV-1 Tat or PMA and iono-
mycin for 1 h, the cells were treated with formaldehyde for chro-
matin-protein cross-linking and lysed, and the cellular DNA was
then purified. DNA-protein complexes were precipitated using
the cross-links were reversed, and the DNA was precipitated and
using five different sets of primers that span the sites of interest
(Fig. 6A) and provide fragments of optimal size for DNA amplifi-
cation for ChIP. By measuring the fold enrichment in amplifica-
tion of the specific antibody immunoprecipitation over the IgG
of interest, we can detect whether the transcription factors under
study bind to that particular region and can compare the binding
HIV-1 Tat, the order of enrichment (from highest to lowest) for
NF-?B was as follows: the ?B2-3/N2-3 position, followed by the
?B1/N1 and then the ?B4/N4 position (Fig. 6B). Thus, NF-?B
appears to preferentially bind to the ?B2-3/N2-3 position in the
HERV-K (HML-2) promoter in response to Tat. Similar results
were obtained for PMA- and ionomycin-induced NF-?B activa-
tion (data not shown). For NF-AT, enrichment is also highest at
the ?B2-3/N2-3 position, followed by the ?B4/N4 position (Fig.
6B). Enrichment levels were of equal proportions when compar-
ing positions ?B1/N1 and N5 (Fig. 6B). No signal was detected
of the region containing the potential Sp1 transcription factor
binding site. qPCR amplification of the Sp1 area never yielded a
Comparing the NF-AT immunoprecipitation to the NF-?B im-
except at the first position (which seems equal to NF-?B enrich-
ment). These data show that there is activation of NF-?B and
NF-AT in response to HIV-1 Tat and that these two transcription
factors interact directly with the HERV-K (HML-2) LTR pro-
We next introduced site-directed mutations into the NF-?B,
struct. We then transfected the mutated HERV-K (HML-2) pro-
moter constructs with or without HIV-1 Tat into Jurkat T cells
and measured the effects on luciferase reporter activity. It should
be noted that some mutations of NF-?B sites also destroy the
potential binding sites for NF-AT proteins, since they were em-
bedded in their DNA sequence. Interestingly, site-specific muta-
ness compared to the wild-type promoter (Fig. 6C). Although
others have reported a role for Sp1 in regulation of the basal ex-
pression of HERV-K (HML-2) (45), mutation of the Sp1 site did
not significantly decrease promoter activity in response to Tat, a
finding consistent with our ChIP data (Fig. 6B). Whereas muta-
tion of the fourth NF-?B/NF-AT (?B4/N4) binding site did not
affect the response to Tat, mutation of the first NF-?B/NF-AT
(?B1/N1) and the second NF-?B (?B2) sites led to a decrease in
crease occurred when the first NF-AT site of the U5 region (N5)
Tat. Mutation of both potential NF-AT sites in the U5 region
simultaneously did not affect the response to Tat any more than
did mutation of the single N5 site (data not shown), suggesting
that the N6 site is not a functional NF-AT binding site, which is
consistent with the ChIP data. Taken together, these data show
that both NF-?B and NF-AT are important for HIV-1 Tat-medi-
scription factor may compensate for loss of the activity of the
other, and that multiple NF-AT and NF-?B binding sites mediate
much of the response to Tat.
Eight percent of the human genome consists of fixed retroviral
elements, termed HERVs, acquired throughout thousands of
nonfunctional, there are some endogenous retroviral genes that
are expressed, even becoming important contributors to human
physiological processes, as can be seen in trophoblast develop-
ment (2, 34, 44, 80). However, HERV gene expression appears to
cellular fitness or integrity is compromised (e.g., with synthetic
chemical agents, radiation, stress, cytokines/chemokines, or bio-
logical agents acting upon human cells) is there pronounced or
increased expression of endogenous retroviral elements (26, 57,
HERV expression can be lifted (130). HIV-1-infected individuals
have abnormally high levels of the endogenous retrovirus
HERV-K (HML-2) expressed in cells, and vastly increased RNA
titers are present in their plasma (24, 25, 27, 50, 130). The conse-
Gonzalez-Hernandez et al.
jvi.asm.org Journal of Virology
putative oncogenes and has been associated with several autoim-
mune, inflammatory, and neurological diseases, it could poten-
tially participate in the development of HIV-1-associated disease
(3, 36, 43, 53, 77, 90, 113). Understanding the initial steps in the
activation of HERV-K (HML-2) expression after HIV-1 infection
is the first step toward deciphering how HERV-K (HML-2) ele-
ments might play a role in HIV-1 pathogenesis.
interact with HERV-K products. The HIV-1 protease, for exam-
actively transport HERV-K RNA from the nucleus to the cyto-
plasm of cells that express it (135). However, these interactions
antibodies specific for NF-?B or NF-AT (or nonspecific IgG isotype controls) 1 h after treatment with either recombinant HIV-1 Tat protein or PMA and
fold enrichment over IgG. Bars are color coded to match primer binding areas. Error bars indicate the SD from three independent immunoprecipitation
type; ?B, NF-?B; N, NF-AT. Luciferase activation was measured 48 h after transfection. Control experiments included a mock transfection and transfection of
an empty vector (pcDNA3.1). Error bars indicate the standard errors of the mean from three independent transfections. Significance was calculated using a
Student t test comparing Tat treatments to vehicle controls or full-length LTR activity to mutant-LTR activity. Significant results are indicated (*, P ? 0.05; **,
P ? 0.005).
HIV-1 Tat Protein Activates HERV-K (HML-2)
August 2012 Volume 86 Number 15jvi.asm.org 7801
likely only partially explain the massive increase in HERV-K
(HML-2) RNA expression seen in HIV-1-infected patients (24,
25, 27). Therefore, we reasoned that another protein from HIV-1
might be involved in stimulating HERV-K (HML-2) expression.
Since the HIV-1 Tat protein has been shown to be a potent acti-
vator of viral (120) and cellular gene expression (17, 108), we
We confirm here that HIV-1 infection leads to increased ex-
pression of HERV-K (HML-2) and show that the HIV-1 Tat and
Vif proteins activate HERV-K (HML-2) expression at the RNA
one HERV-K (HML-2) protein, capsid. In addition, treating pri-
mary lymphocytes (which are more biologically relevant than
continuously passaged cell lines) with HIV-1 Tat also results in
increased HERV-K (HML-2) RNA expression. This effect of Tat
on HERV-K (HML-2) activation was pronounced but dependent
on the cell-type. For cells that constitutively express high levels of
HERV-K (HML-2) transcripts and proteins (such as the terato-
carcinoma cell lines), little response to Tat was detected at the
HERV-K (HML-2) or in primary cells with undetectable or ex-
tremely low levels of transcripts, Tat treatment upregulated tran-
primary lymphocytes, a major target of HIV-1 infection. This ac-
tivation occurred whether Tat was added exogenously to the cells
or if the cells were transfected with constructs encoding it. The
effect occurred at the level of the transcriptional promoter, with
cooperative involvement between Tat and the cellular transcrip-
tion factors NF-?B and NF-AT. Since the promoter also contains
a number of other potential transcription factor binding sites, we
cannot discount the possibility of other factors also contributing
to activation in the presence of Tat. We must note that other
biological agents, chemicals, exogenous infections, or stress con-
ditions can activate HERV expression in cells (39, 55, 66, 68, 94,
activation during HIV-1 infection, it is but one contributor
among the whole spectrum of factors that cause an increase in
HERV expression in a biological system. Interestingly, a recent
study has found that the HTLV-1 Tax protein has been shown to
tors can modulate endogenous retroviral expression.
to verify which HERV-K (HML-2) proteins are expressed at a
higher level in the presence of Tat, but the current status of the
available antibodies precludes quantitatively accurate experi-
in the activation of HERV-K (HML-2) transcription, which helps
to explain the basis for the increased expression of HERV-K
Tat treatment, the degree of stimulation still does not reflect the
ies seen in the plasma of HIV-1-infected patients compared to
also increase production of HERV-K (HML-2). For example, as
HERV-K (HML-2) in patients. Further, a potential cooperativity
between HERV-K (HML-2) Rec and HIV-1 Rev (both of which
can transport HERV-K [HML-2] RNA out of the nucleus) could
lead to increased HERV-K (HML-2) protein expression. Thus,
additional interactions between HIV-1 and HERV-K likely con-
tribute to the marked increase in HERV-K (HML-2) expression
seen in patients with HIV-1 infection.
The consequences of the high levels of HERV-K (HML-2) ex-
pression seen in HIV-1-infected individuals are potentially im-
portant, since increased expression of HERV-K has been associ-
ated with a number of different pathologies (3, 18, 36, 43, 53, 54,
113). HERV-K (HML-2) encodes the Rec protein, a Rev-like pro-
tein that could augment the transport of RNAs out of the nucleus
(14, 78, 135), thus altering the global processing of cellular RNA.
Further, HERV-K (HML-2) encodes two putative oncogenes, rec
and np9 (5, 13, 101), either of which might play a role in HIV-
related malignancies. However, it has recently been shown that
HERV-K antigens promote a T cell response against HIV-1 (50,
123), although Gag- and Env-specific T cell responses are infre-
quent (65). Thus, increased expression of HERV-K (HML-2) fol-
lowing HIV-1 infection might actually be helpful in controlling
the replication of HIV. Further studies need to be conducted in
order to fully ascertain the true impact of HERV-K (HML-2) ac-
tivation in the setting of HIV-1 infection and its contribution to
Fujinaga, Derek Dube, Seagal Teitz-Tennenbaum, and Akira Ono for
their help in experimental design, reagent supplies, and data interpreta-
tion and for their thoughtful comments. The following reagents were
obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH: pNL4-3, pcDNA3.1?/Tat101-flag
(PEV280), pRev-1, pcDNA-HVif, pcDNA-Vphu, pEGFP-Vpr, pHXB2-
env, pcDNA3.1SF2Nef, pMtat(?), clade B recombinant HIV-1 Tat pro-
tein, HIV-1 Tat monoclonal antibody (1D9), the H9 cell line, and the
Jurkat-Tat cell line.
M.J.G-H. was supported by a Rackham Merit Fellowship, the Mecha-
nisms of Microbial Pathogenesis Training Grant from the University of
Michigan, and by an NIH Ruth L. Kirschstein NRSA Individual Predoc-
toral Fellowship to Promote Diversity in Health-Related Research grant
ment as a U.S. Department of Homeland Security (DHS) Graduate Stu-
istered by ORISE, through an interagency agreement with the U.S.
Department of Energy (DOE), contract number DE-AC05-00OR22750.
This research was additionally supported by a generous grant from the
Concerned Parents for AIDS Research and by grants RO1AI062248 and
RO1CA144043 to D.M.M. from the National Institutes of Health.
D.M.M. was also the recipient of a Burroughs Wellcome Fund Clinical
Scientist Award in Translational Research.
All opinions expressed here are the authors’ and do not necessarily
reflect the policies and views of the DHS, DOE, or ORISE.
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