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INTRODUCTION |
Human herpesvirus 8 (HHV8),1 or Kaposi's
sarcoma-associated herpesvirus, is the first human member of
2-herpes viruses (1-3). HHV8 has been identified in Kaposi's
sarcoma (KS) (4-8), primary effusion lymphoma (also termed body
cavity-based lymphoma or BCBL) (9, 10), and multicentric Castleman's
disease (11). Epidemiological surveys report that the seroprevalence
for HHV8 ranges from 0 to 20% in the general population of the Western
world (12). Recently, HHV8 seropositivity was observed to be increased
in populations exposed to sexually transmitted diseases, such as syphilis or human immunodeficiency virus (HIV), suggesting that HHV8 is
also a sexually transmitted pathogen (13). As the etiologic agent for
KS, HHV8 generally results in latent infection in its natural host
cells (14). Based on several extensive studies on the association of
HIV and the development of KS, it is now understood that HHV8 is
required but not necessarily sufficient for the development of KS (15).
In fact, the incidence rate of KS among HIV-1-infected individuals is
up to 100,000-fold higher than that among the general population, and
300-fold higher than that for persons with other types of acquired
immunosuppression. These findings suggest that immunosuppression likely
plays a role as a cofactor with HHV8 in the development of KS; however,
they also suggest that HIV-1 infection, independent of its
immunosuppressive effects, contributes to HHV8-induced KS (16).
Accordingly, it has been proposed by several investigators (17-19)
that the role of HIV-1 in KS potentially involves two additional
events: HIV-1-induced cytokines production and production of HIV-1 Tat
protein. Indeed, cytokines produced by HIV-1-infected cells can induce
lytic cycle replication of HHV8, and Tat can activate the vascular
epithelial growth factor receptor KDR in endothelial cells
(20).
Currently, it cannot be excluded that there may be many yet
characterized interactions between HHV8 and HIV-1 that explain the
dramatically higher incidence of KS in coinfected individuals. Thus,
additional HIV proteins and/or cellular factors induced by HIV-1 could
contribute to KS pathogenesis with possibly complex reciprocal effects
between HHV8 and HIV-1. Consistent with these thoughts, many
herpesviruses such as Epstein-Barr virus (21, 22), cytomegalovirus
(23-25), human herpesvirus 6 (26-28), and human herpesvirus 7 (29,
30) have been described to influence HIV-1 replication and expression.
To decipher mechanistic interplays between HHV8 and HIV-1, several
relevant findings need to be considered. Typically, HHV8 is found in B
lymphocytes, keratinocytes, epithelial cells, KS tumor cells, and
endothelial cells (31). By contrast, the predominant host cells for
HIV-1 are CD4+ T lymphocytes, dendritic cells, and mononuclear
phagocytes (32, 33). Interestingly, Moir and colleagues (34) have shown
recently that the up-regulation of CD4 and CXCR4 on B lymphocytes
mediated by CD40 stimulation leads to increased susceptibility of these
B lymphocytes to T-tropic HIV-1 infection. Conversely, dendritic cells
and macrophages have been found to be susceptible cells for HHV8
infection (35, 36). Additionally, HHV8-infected B lymphocytes are
further infectable by HIV-1 through a cell-cell-mediated pathway (37).
Therefore, it stands to reason that in vivo HHV8 and HIV-1
genomes could coexist in some cells in dually infected individuals.
With the goal of elucidating better intracellular interactions between
HHV8 and HIV-1, we queried for potential reciprocal effects on gene
expression between HHV8 and HIV-1. We found that when the HHV8-positive
primary effusion lymphoma cell line BCBL-1 (38) was fused to
HIV-1-latent ACH2 cells (39, 40), enhanced expression was observed from
both HHV8 and HIV-1 genomes. Further analyses indicated that sole
expression of the HHV8 KIE2 (41) protein was sufficient to activate the
HIV-1 LTR. On the other hand, expression of either HIV Vpr (42) or Tat
(43) was sufficient to modulate HHV8 expression. Our data suggest that
reciprocal transcriptional activation between HHV8 and HIV-1 may
explain in part the immunosuppression-independent contribution of HIV-1 to HHV8-induced KS.
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EXPERIMENTAL PROCEDURES |
Cells and Culture Conditions--
BCBL-1 and ACH2 were obtained
from the National Institutes of Health AIDS Research Reagent and
Reference Program. Jurkat cells were purchased from the American Type
Culture Collection. BCBL-1, ACH2, and Jurkat cells were grown in RPMI
1640 supplemented with 10% heat-inactivated fetal bovine serum at
37 °C in the presence of 5% CO2. HeLa cells were
maintained in Dulbecco's modified Eagle medium supplemented with 10%
heat-inactivated fetal bovine serum. For induction of HIV expression in
ACH2 cells, 50 ng/ml TPA and 2 µg/ml PHA were added into complete
medium (44). BCBL-1 cells were treated with 20 ng/ml TPA for 3 days to
stimulate HHV8 expression (2).
Cell-Cell Fusion--
HIV latent ACH2 cells were mixed with
BCBL-1 in equal number. After washing twice with phosphate-buffered
saline, cells were resuspended into 1 ml of 100% (w/v) polyethylene
glycol 4000 in RPMI 1640 containing 5% (v/v) dimethyl sulfoxide. The
cell/polyethylene glycol suspension was centrifuged at 100 × g for 2 min, thereafter, 10 ml of RPMI 1640 were added to
resuspend the cell pellet. Cells were then centrifuged at 100 × g for 5 min, supernatant was removed, and cells were finally
resuspended into complete medium (46).
RNA Preparation and Ribonuclease Protection Assay--
Total RNA
was isolated form fused cells with TRIzol reagent (Life Technologies,
Inc.) according to the manufacturer's instructions. RNA (50 µg) was
hybridized with the 32P-labeled antisense RNA probe (3 × 105cpm). Hybridized reactions were digested with RNase A
and RNase T1, and the nuclease-protected RNA probe was analyzed on a
5% denaturing acrylamide gel containing 7 M urea. HIV-1
antisense TAR probe was constructed as described previously (47).
Antisense probe to the HHV8 IL-6 sequence (GenBankTM accession no.
U93872) from nucleotide 17242 to 17279 corresponding to the
vIL-6 coding region was transcribed from the Sp6 promoter in
the pGEM IL-6 plasmid, which contains a cloned IL-6 sequence. Antisense
actin probe was transcribed using T7 RNA polymerase from vector
pTRI-
-actin-human (Ambion, Austin, TX). Protected actin transcript
corresponds to a band of 245 bp.
Constructions of Wild-type and Mutant HIV-1 LTR--
The
luciferase cDNA under the control of the HIV-LTR (pLTR) was
constructed in the pGL2 basic vector. HIV-LTR mutant reporters were as
follow: pLSM1 with
111/
94 deletion, pLSM2 with
93/
79 deletion,
pLSM3 with
105/
81 deletion, pLSM4 with
75/
58 deletion, and
pLSM5 lacking of
57/
40 nucleotides. These luciferase reporter plasmids were from the NIAID AIDS Reagent Repository.
Transfection of Plasmids--
The promoter activities of
wild-type and mutant HIV LTR were assayed by transiently transfecting
BCBL-1 and HeLa cells, which were either mock-treated or treated with
TPA for 48 h prior to the transfection. To analyze the
transcriptional effects of HHV8 immediate-early genes on HIV LTR,
Jurkat cells were transfected with KIE expression vectors and cognate
reporter constructs. Amounts of transfected DNA were equalized using
pCMV Tag2B. Each transfection also included a cotransfected pCMV
-galactosidase plasmid whose activity was used to normalize
for transfection efficiency. 5 × 105 Jurkat cells
were transfected using LipofectAMINE 2000 (Life Technologies, Inc.) in
a 24-well plate. All transfection reactions were performed in
triplicate; values were calculated for standard deviations.
Alternatively, BCBL-1 cells (1 × 107) were placed in
0.5 ml of electroporation buffer (RPMI 1640 containing 15% fetal
bovine serum, 1 mM sodium pyruvate, 2 mM
L-glutamine, 1× nonessential amino acids) (48) and were
mixed with 10 µg of either Vpr or Tat expression vector. Cells were
then electroporated at 250 V and 960 microfarads using a Gene Pulser
(Bio-Rad Laboratories, Hercules, CA). After pulse application, cells
were immediately pelleted by centrifugation and then kept for 20 min at
room temperature (49). Transfected cells were cultured in RPMI 1640 supplemented with 20% heated-inactivated fetal bovine serum. The
efficiency of transfection was measured by transfecting BCBL-1 with
plasmid pEGFP-C (CLONTECH Laboratories, Palo Alto,
CA), which contains the enhanced green fluorescence protein gene fused
to the CMV immediate-early gene promoter. After 24 h, 40% of the
electroporated cells routinely displayed green fluorescence.
Cloning of HHV8 KIE cDNAs--
BCBL-1 cells, which
had been chemically induced into lytic cycles, were harvested after
exposure to 3 mM sodium butyrate for 4 h.
Poly(A)+ RNA was isolated with a QuickPrep Micro mRNA
purification kit (Amersham Pharmacia Biotech, Piscataway, NJ) and was
reverse-transcribed using a Fast-Run RT-PCR kit (Protech, Taiwan). The
cDNA sequences of HHV8 immediate-early genes KIE2,
KIE3, and KIE1 type I (41) were based on
GenBankTM accession nos. AF091346, AF091347, and AF091348,
respectively. The KIE1 gene of HHV8 transcribes three
splicing variants ranging from 3.6 to 3.8 kb in length. These species
encode three putative open reading frames ORF50, ORFk8.2, and ORFk8
.
cDNAs for ORF50, ORFk8.2, and ORFk8
were also cloned separately
into expression vectors. The following oligonucleotide primers were
used in RT-PCRs: 5'-end of KIE2, 5'-GCGCAAGCTTATGGCGATGTTTGTG-3'; 3'-end of
KIE2,
5'-GCATCTCGAGTTATCAGTCCAGCCA-3'; 5'-end of
KIE3,
5'-CGCCGATATCATGCAAATTAGCTTT-3'; 3'-end of
KIE3,
5'-ATATCTCGAGTTATTGAAGCCCAGG-3'; 5'-end of
KIE1-50, 5'-GGGTGATATCATGGCGCAAGATGAC-3'; 3'-end of
KIE1-50,
5'-GGTACTCGAGTCAGTCTCGGAAGTA-3'; 5'-end of
KIE1-8.2,
5'-GCCGGATATCATGTTGAAGCTTGGT-3'; 3'-end of
KIE1-8.2,
5'-GGATCTCGAGCTATGTAGGGTTTCT-3'; 5'-end of KIE1-8
,
5'-CCGCGATATCATGCCCAGAATGAAG-3'; 3'end of
KIE1-8
, 5'-GCAACTCGAGTCAACATGGTGGGAG. Recognition sites
for restriction enzymes are underlined; boldface
nucleotides denote the start (5'-end oligonucleotides) and stop
codons (3'-end oligonucleotides). PCR conditions for KIE2
and KIE1-8
cDNA amplification were 35 cycles at
94 °C, 1 min; 53 °C, 1 min; and 72 °C, 1 min and that for
KIE3, KIE1-50, and KIE1-8.2 were 35 cycles at 94 °C, 1 min; 50 °C, 1 min; and 72 °C, 1.5 min. The
PCR-generated fragments of the HHV8 KIE cDNAs were
subcloned into pCMV Tag2B (Promega, Madison, WI) to yield flag-tagged
pKIE2, pKIE3, pKIE1-50, pKIE1-8.2, and pKIE1-8
.
Reporter Assay--
Cells were harvested 72 h
post-transfection and were resuspended into lysis buffer (0.5 M HEPES, 0.2% Triton X-100, 1 mM
MgCl2, 1 mM CaCl2, pH 7.8). After
three cycles of rapid freezing and thawing, cell extracts were obtained
by centrifugation at 14,000 rpm for 5 min at 4 °C and were analyzed
for luciferase activity using a Luciferase assay system (Packard,
Meriden, CT).
-Galactosidase assays were performed by incubating
cell extracts with an equal volume of
-galactosidase assay buffer
(Packard). Readout values of luciferase activity were normalized
against corresponding
-galactosidase value for each sample within
each experiment.
RT-PCR--
Total RNA (1 µg) was reverse-transcribed using a
1st Strand cDNA synthesis kit for RT-PCR (Roche Molecular
Biochemicals, Indianapolis, IN). To ensure that no DNA contaminated the
isolated RNA, the RNA samples were treated with DNase I (Stratagene, La
Jolla, CA) prior to reverse transcription. The mixtures containing
random hexamers for cDNA syntheses were incubated at 25 °C for
10 min and then at 42 °C for 1 h. By heating at 95 °C for 5 min, the avian myeloblastosis virus reverse transcriptase was
denatured. As an additional control, each sample was also subjected to
reverse transcription in the absence of RT. Two microliters of the
cDNA products were then added to 48 µl of PCR reaction mixture,
which contained HHV8 major capsid protein primers (forward:
5'-GTACACCTATTTCTTCCCTGTTGG-3'; reverse: 5'-TGCTCATACCTGAGACCGTACC-3')
and internal control 18 S rRNA primers/competitors (Ambion,
Austin, TX) at a 2:8 ratio. The 18 S rRNA competitors were modified at
their 3'-ends to block extension by DNA polymerase. Amplification was
carried out according the following protocol: preheating at 95 °C
for 5 min followed by 25 cycles of 95 °C, 58 °C, and 72 °C for
30 s in each segment. The PCR products of major capsid protein
(270 bp) and 18 S rRNA (314 bp) were analyzed on a 2.5% agarose gel.
To obtain meaningful results, the linear range of relative quantitative
RT-PCR was determined by graphing cycles versus products,
which were quantitated with an Alpha Innotech Corp. IS-1000 digital
imaging system. The amplification was found to be in a linear range of
20 to 29 thermal cycles.
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RESULTS |
Reciprocal Activation of Transcription between HHV8 and
HIV-1--
To better understand functional interactions between HHV8
and HIV-1, a series of cell fusion experiments was done. We took advantage of the fact that ACH2 and BCBL cells are latently infected with HIV-1 and HHV8, respectively. Both genomes can be activated from
latency by treatment of cells with phorbol ester, TPA. By fusing
together ACH2 and BCBL cells, heterokaryons can be created, which simultaneously contain both viral genomes. In this manner, potential interactions between the two viruses in the same cell could
be assayed. When ACH2 cells were fused with TPA-induced BCBL-1 cells,
RNase protection assay showed that HIV-1 TAR-containing RNAs were
markedly increased when compared with control samples (Fig.
1A). Because all HIV-1
transcripts contain TAR sequence, this finding suggests that some
element associated with the HHV8 genome could activate transcription
from the HIV-1 LTR. Indeed, the BCBL-1-mediated transcriptional
enhancement was considerably stronger than that achieved by chemically
inducing ACH2 cells with TPA and PHA. Thus the observed effect is
likely a functional consequence of an HHV8 gene product and not a
trivial result of TPA carryover from the induced BCBL-1 cells.
Conversely, in a reciprocal experiment, when HHV8 gene expression was
analyzed, the level of vIL-6 RNA was increased significantly when
BCBL-1 cells were fused with TPA-induced ACH2 cells (Fig.
1B).

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Fig. 1.
RNase protection assay (RPA) of the
expression of HHV8 and HIV-1in cell-cell fusions of ACH2 and BCBL-1
cells. A, 50 µg of total RNA was isolated from
HIV-latently-infected ACH2 cells either without or with chemical
(TPA/PHA) induction (lanes 1, 2) or
HHV8-harboring BCBL-1 cells either without or with TPA induction prior
to cell fusion (lanes 3, 4). RNAs were then
incubated with 3 × 105 cpm of antisense HIV TAR
(upper panel) and human -actin probes (lower
panel) in RPA. B, RPA of total RNA (50 µg) from
BCBL-1 cells either without or with TPA induction (lanes 1,
2) and ACH2 cells either without or with chemical induction
prior to fusion with BCBL-1 (lanes 3, 4). RNA
expression was quantitated using antisense HHV8 VIL-6. Protection of
human -action mRNA was used as a normalizing control. Size
markers are indicated at the left of the panel. The ratio of
integrated density values (IDV) of target signal against
-action is shown under the RPA results.
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KIE2 Gene of HHV8 Activates HIV-1 Transcription Synergistically
with Tat--
We next attempted to define the HHV8 responsive element
within the HIV-1 LTR. Six scanning mutants that abrogated separately the NF-
B sites, the Sp1 sites, or the TATA element were utilized (Fig. 2A). Although the basal
activities of each of the mutant promoters were different when each was
transfected into TPA-stimulated BCBL-1 cells, similar levels of
HHV8-associated induction were observed. These results indicate that
HHV8-responsiveness involves complex, rather than simple sequence
motif(s). Control transfection of the same mutant promoters in HeLa
cells (Fig. 2B) with or without TPA failed to show
comparable activation consistent with activation being specific for
BCBL-1-associated HHV8 gene product(s).

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Fig. 2.
Expression of wild-type HIV-1 LTR and
LTR-mutants in TPA-treated BCBL-1 and HeLa cells. A,
schematic representations of reporter plasmids used in the
transfections. Wild-type and the indicated mutants of the HIV-1 LTR
(0.4 µg) were transfected into BCBL-1 (B) or HeLa cells
(C) with or without prior TPA treatment. Promoter activities
(measured by luciferase readouts) obtained 48 h post-transfection
after normalization for -galactosidase are expressed relative to
that of pLTR-luc in cells without induction, which was arbitrarily set
as 100 units. CPS, counts per second.
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To further clarify how HHV8 activates HIV-1, we sought to identify
responsible viral gene product(s). Although current understanding of
HHV8 gene functions remains incomplete, we reasoned, based on analogy
to other herpesviruses, that immediate-early genes of HHV8 are
plausible candidate activators of HIV-1. Hence, we cloned five
immediate-early open-reading frames (ORFs) from HHV8 and tested for
their effects on the HIV-1 LTR. Our results showed that out of these
five ORFs, only KIE2 activated the HIV-1 LTR (Fig.
3A). To check whether
activation by KIE2 of HIV-1 LTR correlates with that observed in BCBL-1
cells (Fig. 2) we tested the series of linker-scanning LTR mutants used
above. Similar to the activation profiles in Fig. 2, KIE2 activated
each linker-scanning mutant comparably (Fig. 3B).

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Fig. 3.
HHV8 KIE2 activates HIV-1 LTR.
A, Jurkat cells were transfected with 0.2 µg of HHV8
KIE expression vectors and 0.1 µg of pLTR-luc.
B, Jurkat cells were transfected with 0.1 µg of pKIE2 and
0.1 µg of wild type HIV-1 LTR or LTR mutants. Cells were harvested
72 h post-transfection and normalized for -galactosidase. The
activity of pLTR-luc alone was arbitrarily set as 100.
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Because the magnitude of activation by KIE2 alone was less than that
observed in the ACH2-BCBL-1 fusion experiments (Fig. 1) we considered
whether there could be a synergy between this HHV8 protein and the
HIV-1 transactivator, Tat. Indeed, when KIE2 and Tat were coexpressed,
a significantly synergistic activation was observed for the HIV-1 LTR
(Fig. 4). Activation reached a level
comparable to that observed in the cell-cell fusions (Fig. 1).

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Fig. 4.
KIE2 acts synergistically with Tat on the
HIV-1 LTR. Jurkat cells were transfected with KIE2,
Tat, or both, together with 0.1 µg of pLTR-luc. Cell
lysates were harvested 72 h post-transfection for luciferase
assay. Numbers on the top of the bars represent
the -fold activation over the basal HIV-1 LTR promoter. The results in
each figure are from one experiment performed in triplicates ± S.D.
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HIV-1 Tat and Vpr Are Activators of HHV8 Expression--
The HIV-1
genome encodes two transcriptional transactivators, Tat and Vpr. We
reasoned that Tat and/or Vpr might account for HHV8 activation by
HIV-1. Using LipofectAMINE, we could achieve only a 10-20%
transfection efficiency of BCBL-1 cells. On the other hand, using
electroporation we routinely transfected up to 40% of BCBL-1 cells.
When BCBL-1 cells were electroporated with expression vectors
expressing either Vpr or Tat (Fig. 5) a
quantitative RT-PCR assay revealed highly enhanced expression of HHV8
major capsid protein (50). Thus, functionally, both Vpr and Tat appear
to be HIV-1 transcriptional activating counterparts of HHV8 KIE2.

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Fig. 5.
Expression of major capsid protein is
stimulated by HIV-1 protein Vpr and Tat in BCBL-1 cells. At
72 h post-transfection, RNAs from BCBL-1 cells transfected with
Vpr or Tat expression vectors were isolated for RT-PCR. The PCR
products of major capsid protein (270 bp) and 18 S rRNA (314 bp) were
analyzed on a 2.5% agarose gel.
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DISCUSSION |
HHV8 is a large (~170 kbp) double-stranded DNA herpesvirus (51).
HIV-1 is a much smaller (~10 kbp) retrovirus. Despite rather dramatic
molecular dissimilarities between the two viral genomes, recent
findings have provided compelling evidence that these two viruses
interact to promote KS pathogenesis in dually infected individuals.
Indeed, it is increasingly clear that, although the HHV8 genome is
invariably present in all described cases of KS (6, 8), most
individuals who are singularly infected with HHV8 carry the virus in a
latent state and do not present with KS disease. On the other hand,
persons infected with both HHV8 and HIV-1 have up to a 100,000-fold
increase, compared with infection with HHV8 alone, in risk for
developing KS (16). Based on this statistic, a reasonable deduction is
that HHV8 is necessary but insufficient for producing KS and that HIV-1
is an important cofactor, which promotes HHV8 induced KS.
How might HIV-1 contribute to the development of KS? The results from a
large multicenter study on HHV8 and HIV-1 infected men established an
unmistakable link between HIV-1-associated immunosuppression and
HHV8-engendered KS (52). However, in the same study it is interesting
that when immunosuppression was excluded, an independent association
was revealed between HIV-1 RNA expression and KS progression (52). The
latter observation provided the impetus for us to systematically
explore potentially more direct interactions between HHV8 and
HIV-1.
Efforts to identify more direct interactions between HHV8 and HIV-1
have, to date, centered largely on HIV-1-induced cytokines and the
secretion of Tat protein from HIV-1-infected cells (53, 54).
Intracellular interactions between the two viruses have been
generally discounted, because a prevailing view has been that the two
viruses infect different cell types. However, several points argue that
such an assumption might not necessarily be warranted. First, it
remains unclear as to what are the range of HHV8 susceptible cells
in vivo. Second, recent ex vivo infection studies
clearly support that both HHV8 and HIV-1 can efficiently infect cells
of the monocyte/macrophage lineage (35, 36). Finally, Spearman and
colleagues (37) have intriguingly demonstrated that HHV8-genome
containing B lymphocytes, in fact, can be infected by HHV-1 via a
cell-cell pathway and that such infected B lymphocytes could support
productive HIV-1 replication.
Here we have tried to define which virally encoded component of HIV-1
and HHV8 might reciprocally influence each other's expression inside
cells. Our cell fusion results (Fig. 1) indicate that when both genomes
exist in the same cellular environment there is a bilateral
transcriptional effect between the two viruses. Among the many ORFs
that are encoded by HHV8, we found that the KIE2 protein was sufficient
for singularly inducing transcription from the HIV-1 LTR (Fig. 3). On
the other hand, two HIV-1-encoded factors, Tat and Vpr, were capable of
individually activating HHV8 transcription (Fig. 5). Although Tat has
previously been invoked to affect HHV8 expression (55, 56), the finding
here that Vpr provides a similar activity adds further complexity to
the interplay between HIV-1 and HHV8.
The concept of reciprocal interaction between HHV8 and HIV-1
potentially offers an explanation for the rapid development of KS in
HHV8-infected individuals whom are subsequently infected with HIV-1.
Thus, in this dually infected subpopulation, the risk for KS
development/progression escalates quickly, with a 60% increase for
each year of HIV-1 infection (52). If, as suggested here, HIV-1
infection might transcriptionally activate HHV8 genome from latency,
and, if such activation of HHV8 leads to further amplification of HIV-1
transcription, then one could imagine how such a feedback-cycle might
accelerate disease manifestation. Indeed consistent with this scenario,
an interruption of this cycle through treating HIV-1 with reverse
transcriptase (RT) or protease inhibitors has been shown to
efficaciously induce the regression of KS (45). Future investigation
toward understanding how to further intervene against the molecular
interplay between HHV8 and HIV-1 should usefully advance the treatment
of KS.