Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein
Binds and Protects a Nuclear Noncoding RNA from
Cellular RNA Decay Pathways
Brooke B. Sahin, Denish Patel, Nicholas K. Conrad*
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated
through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the
integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable
of destroying viral transcripts. Here we report that the Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 protein binds
to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57
increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse
assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on
transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments,
respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells
and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is
necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless b-globin
mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them
from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of
intronless viral transcripts by protecting them from a cellular RNA quality control pathway.
Citation: Sahin BB, Patel D, Conrad NK (2010) Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein Binds and Protects a Nuclear Noncoding RNA from Cellular
RNA Decay Pathways. PLoS Pathog 6(3): e1000799. doi:10.1371/journal.ppat.1000799
Editor: Jae U. Jung, University of Southern California School of Medicine, United States of America
Received November 2, 2009; Accepted January 28, 2010; Published March 5, 2010
Copyright: ? 2010 Sahin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NKC is a Southwestern Medical Foundation Scholar in Biomedical Research. This work was funded by the American Cancer Society (ACS-IRG 02-196)
and the Southwestern Medical Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Post-transcriptional events in mRNA biogenesis are of central
importance to the fidelity and regulation of gene expression.
Cellular factors regulate nearly every step of RNA metabolism
including transcription elongation, RNA splicing, 39 end forma-
tion, nuclear export, translation, etc. In fact, genome-wide
profiling experiments demonstrate that a significant percent of
the observed changes in RNA levels are dictated by regulation of
the stability of a transcript rather than its transcription (e.g. [1,2]).
RNA half-life can be modulated directly, through the activities of
regulatory stabilizing or destabilizing protein factors or small
RNAs [3–5]. In addition, RNA quality control pathways ensure
aberrant transcripts are less stable than their functional counter-
parts . Given the importance of these pathways for gene
expression, it is no surprise that viruses have evolved mechanisms
to counteract pathways that otherwise would lead to the
destruction of viral transcripts [3,7].
The Kaposi’s sarcoma-associated herpesvirus (KSHV) is a
member of the gammaherpesvirus family that causes Kaposi’s
sarcoma, a common AIDS-associated malignancy, as well as the
lymphoproliferative disorders primary effusion lymphoma (PEL)
and some cases of multicentric Castleman’s disease (MCD) [8–10].
The life cycle of KSHV includes a latent phase in which the viral
DNA is maintained in infected host cells as a circular episome.
During latency, few viral genes are expressed and no viral
replication occurs. When the KSHV lytic phase is reactivated, a
well-regulated cascade of gene expression is initiated by the viral
transactivator ORF50 (Rta) resulting in infectious virus production
[11–13]. Like all herpesviruses, the KSHV genome is nuclear and
its genes are expressed utilizing the host cell transcription, RNA
processing, and translation machinery. In many respects, KSHV
genes resemble those of their host; that is, they have canonical
promoter elements, 39-end formation signals, and consensus pre-
mRNA splice sites.
However, KSHV genes differ from canonical cellular genes in
several relevant ways. Some transcripts are bicistronic, KSHV
introns are smaller than the average size of a mammalian intron,
and genes are more closely arranged in the genome than host
genes. Most importantly for the present work, ,70% of KSHV
genes lack introns , whereas most human protein-coding genes
contain multiple introns . This difference in gene structure has
implications for the expression of viral genes. The presence of an
intron in a pre-mRNA and/or the changes in ribonucleoprotein
particle (RNP) composition that result from splicing promote the
efficiency of almost every stage of gene expression, including
PLoS Pathogens | www.plospathogens.org1 March 2010 | Volume 6 | Issue 3 | e1000799
transcription initiation and elongation, 39-end formation, mRNA
export, RNA localization, and translation [16–27]. As a result,
transgenes containing an intron are often expressed at significantly
higher levels than those same genes lacking an intron . To
compensate for the lack of introns or splicing, viruses that express
unspliced or intronless transcripts have evolved mechanisms that
promote efficient gene expression in the absence of splicing
KSHV encodes a viral post-transcriptional regulator of gene
expression called ORF57 (Mta, KS-SM) that is essential for viral
replication [34,35]. ORF57 is a member of a conserved family of
herpesvirus proteins that post-transcriptionally enhance gene
expression [29–33]. ORF57 has been implicated in a variety of
steps of RNA biogenesis from transcription to translation and it
increases the efficiency of intronless gene expression [30,31,36,37].
ORF57 has been reported to interact with ORF50 and to enhance
transcription in a promoter and cell-type specific manner [37–39].
In addition, ORF57 binds cellular export factors and promotes the
nuclear export of at least a subset of intronless viral mRNAs
[40–43]. Unlike the herpes simplex homolog (HSV) ICP27, which
contributes to host gene shut-off by inhibiting splicing, ORF57
promotes the splicing of some viral mRNAs [35,44], and splicing
activity is also seen with the Epstein-Barr virus (EBV) ORF57
homolog, SM . ORF57 has further been suggested to play a
role in translation of an internal ribosome entry site-containing
reporter . Thus, ORF57 is a multifunctional regulator of
mRNA biogenesis that may, in part, compensate for the lack of
introns in viral gene expression.
ORF57 is critical for the accumulation of the polyadenylated
nuclear (PAN) RNA (nut1, T1.1) [34,35,37,42], a non-coding
nuclear transcript that accumulates to high levels during the lytic
phase of viral infection [47,48]. The PAN RNA promoter is
ORF50-dependent [49,50], and PAN RNA accumulation further
depends on the activity of a 79-nucleotide (nt) RNA element,
called the ENE [51–53]. Mechanistically, the ENE interacts in cis
with the poly(A) tail of PAN RNA resulting in the sequestration of
the poly(A) tail from exonucleases. Detailed kinetic analysis of the
effects of the ENE on PAN RNA decay in transfected cells showed
that PAN RNA is subject to two kinetically distinguishable decay
pathways, one with a very short half-life (10–20 min) and another
with a longer half-life (3–5 hrs). ENE-lacking or ENE-mutant PAN
transcripts are more likely to be degraded in the rapid RNA decay
pathway than are their wild-type ENE containing counterparts.
Because the ENE is sufficient to increase the nuclear accumulation
of heterologous intronless transcripts, we further proposed that this
rapid decay pathway is part of a nuclear RNA surveillance system
that rapidly degrades inefficiently exported mRNAs.
ORF57-mediated enhancement of the exclusively nuclear PAN
RNA suggests that it may be involved in inhibiting the proposed
RNA surveillance mechanism. Here, we test this idea and find that
ORF57 stabilizes PAN RNA, particularly those transcripts that
lack the ENE. We see no ORF57-dependent effect on RNA
polymerase II (pol II) density on the PAN RNA gene nor does
ORF57 lead to PAN RNA export. Therefore, we conclude that
the observed stability enhancement constitutes the major effect of
ORF57 on PAN RNA accumulation. In addition, ORF57 binds
PAN RNA directly in vivo and a deletion of the 59 portion of PAN
RNA, dubbed the ORF57-responsive element (ORE), reduces
ORF57 binding and ORF57 response. We show that tethering of
ORF57 to an ORE-deleted PAN RNA restores ORF57-mediated
up-regulation. Finally, we show that the ORE is sufficient to confer
increased ORF57-response to a heterologous intronless b-globin
mRNA, but not its spliced counterpart. We conclude that ORF57
protects viral transcripts from the same cellular RNA decay
pathway that the ENE protects from in cis and that its stabilization
activity is dependent on ORF57 binding to target RNAs.
ORF57 protects transcripts from rapid decay
If ORF57 protects transcripts from RNA decay pathways in
vivo, we reasoned that the effects of ORF57 would be more
pronounced on unstable ENE-lacking transcripts than on their
ENE-containing counterparts. To test this idea, we compared the
RNA levels of PAN RNA containing the ENE (PAN-WT) to PAN
RNA lacking the ENE (PAN-D79) in the absence of ORF57 or in
its presence. We transfected HEK293 cells with constructs that
express PAN-WT or PAN-D79 and co-transfected ORF57-
expression constructs at two concentrations or empty vector. After
,18-24 hours, we extracted total RNA, and quantified relative
RNA levels by northern blot (Figure 1). Consistent with published
results [34,35,37,42], ORF57 increases wild-type PAN RNA levels
in a dose-dependent fashion (Figure 1A, lanes 1–3). Quantitation
of these data show that, at the highest ORF57 concentration
tested, PAN RNA is ,3.4-fold more abundant (Figure 1B). As
predicted from our model, ENE-lacking transcripts show an even
greater response to ORF57, ,11-fold (lanes 4–6 and Figure 1B).
Because the ENE is involved in RNA stability, these results are
consistent with the conclusion that ORF57 increases the half-life of
To directly examine the effects of ORF57 on PAN RNA half-
life, we employed a transcription pulse strategy . In these
experiments, we transfected HEK293 Tet-off advanced (293TOA)
cells with TRP-D79, a plasmid that expresses the ENE-lacking
PAN-D79 transcript from a tetracycline-responsive promoter .
In 293TOA cells, transcription from this promoter is turned off in
the presence of doxycycline (dox, a tetracycline analog), and is
induced in its absence. In our initial experiments, we examined the
effects of ORF57 on PAN RNA decay after a two-hour
transcription pulse (Figure 2). As expected, TRP-D79 RNA was
undetectable prior to transcription pulse, but can be detected after
In order to replicate efficiently, a virus must ensure that its
genes are properly expressed in the context of an infected
host cell. Recent work has demonstrated that eukaryotic
cells have RNA quality control pathways that degrade
improperly processed, aberrant RNAs. Our published
findings using an unusual Kaposi’s sarcoma-associated
herpesvirus (KSHV) nuclear RNA, called PAN RNA, have
suggested that intronless polyadenylated transcripts are
subject to such a quality control system. Because most
KSHV genes lack introns, we hypothesized that KSHV must
have evolved mechanisms that bypass this quality control
system. In support of this idea, we show that the ORF57
protein, a multifunctional enhancer of KSHV gene expres-
sion, binds to and stabilizes PAN RNA. We further define an
element called the ORF57-responsive element (ORE) in
PAN RNA that is necessary for ORF57-binding and activity
on PAN RNA. In addition, we show that the ORE is
sufficient to confer ORF57-responsiveness to a heterolo-
gous intronless mRNA, but not its spliced counterpart.
These observations substantiate the model that ORF57
enhances KSHV gene expression by protecting viral
transcripts from host RNA surveillance pathways. More
broadly, these data suggest that viruses producing
intronless nuclear RNAs require mechanisms to evade
host quality control mechanisms.
KSHV ORF57 Binds and Stabilizes PAN RNA
PLoS Pathogens | www.plospathogens.org2March 2010 | Volume 6 | Issue 3 | e1000799
two hours in dox-free media (Figure 2A, top panels). Examination
of the decay profiles clearly shows an increase in RNA stability
when ORF57 is expressed (Figure 2B, top). Interestingly, the
mobility of a portion of remaining transcripts after transcription
shut-off is reduced while others show increased mobility. We have
determined that these mobility changes are due to differences in
poly(A) tail length (data not shown). The relationship between
changes in poly(A) length and ORF57 function is currently under
investigation and will be described elsewhere (see Discussion). We
also examined the effects of ORF57 on TRP-WT, a PAN
expression construct containing the ENE . However, in
293TOA cells this plasmid produced an extremely stable
transcript (t1/2.24hr), impractical for use in decay assays. Overall,
these data demonstrate that ORF57 increases the half-life of
unstable PAN RNA transcripts.
Previous studies showed that PAN RNA is subject to two decay
pathways with different kinetic properties [51,52]. That is, one
pool of PAN RNA transcripts is degraded very rapidly with half-
lives of ,10-20 minutes, while another pool of transcripts is
degraded more slowly (t1/2,3-5hrs). The presence of the ENE
appears to protect transcripts from the rapid decay system
resulting in a decrease in the fraction of transcripts that are
observed in this population. Consistent with these published
findings, the decay profiles in Figure 2B are nicely fit by two-
component exponential decay curves where the two components
represent the two pools of transcripts. Using regression analysis, we
can determine the decay parameters in PAN RNA degradation,
including the fraction of transcripts undergoing rapid decay and
the half-life of each population. Because ENE-lacking transcripts
are preferentially up-regulated by ORF57, we predicted that, like
the ENE, ORF57 expression would decrease the fraction of PAN
transcripts in the rapid RNA decay pathway.
To test this idea, we performed regression analysis of the data
for TRP-D79 RNA decay in the presence or absence of ORF57
(Figure 2B, Table S1 and Figure S1). Examination of the kinetic
parameters shows that in the absence of ORF57, 73% of the
transcripts are in the rapidly degrading population (t1/2,7.8 min)
(Figure 2C). In contrast, only 51% of the transcripts are degraded
rapidly when ORF57 is co-expressed, a statistically significant
decrease. Because the more slowly degrading transcripts accumu-
late over time, the fraction of transcripts observed in the rapidly
degrading pool decreases when longer transcription pulse times
are employed . If ORF57 decreases the fraction of transcripts
that degrade rapidly, it follows that the observed rapidly degrading
fraction would decrease more quickly when ORF57 is present.
Indeed, the effects of ORF57 on TRP-D79 RNA decay are even
more apparent after an 18-hour transcription pulse (Figure 2A and
Figure 2B). In this case, the apparent half-life (i.e. the time
difference at 50% remaining, Figure 2B) is increased ,8-fold.
More importantly, the percent of transcripts degrading rapidly in
the presence of ORF57 is reduced to 15%, while 57% is rapidly
degraded in its absence (Figure 2C). Taken together, these data
strongly argue that ORF57 enhances PAN RNA levels by
protecting it from a rapid cellular RNA decay pathway.
ORF57 up-regulation of PAN RNA occurs after
Previous reports suggested that ORF57 enhances transcription
rates of specific promoters in certain cell types, including the PAN
RNA promoter in 293 cells [37–39]. Even though we see an
increase in PAN RNA half-life in the presence of ORF57, it
remains possible that a significant portion of the up-regulation of
PAN RNA by ORF57 is at the level of RNA synthesis rather than
decay. To test the effects of ORF57 on transcription initiation, we
initially examined the response of PAN RNA to ORF57 from
three different promoters (Figure 3A). Consistent with the results
of others, PAN RNA expression from the cytomegalovirus
immediate early (CMVIE) promoter is responsive to ORF57
[36,37,39]. We extended this analysis by examining PAN RNA
steady-state levels driven by the cellular elongation factor 1a
(EF1a) and viral SV40 promoters (Figure 3A). Each of these
constructs is 39 processed using the PAN RNA cleavage and
polyadenylation signals. For every promoter tested, ORF57
increased PAN RNA levels in a dose-dependent fashion
supporting a post-transcriptional role for ORF57. Interestingly,
the magnitude of the change differs among the constructs and this
does not necessarily correlate with the strength of each promoter.
Figure 1. ORF57 preferentially enhances the levels of an
unstable nuclear RNA. (A) Top, schematic diagram of the constructs
used. Both are driven by the PAN promoter (gray) and have the PAN 39-
end formation signals (black). PAN-D79 has the 79-nt ENE sequence
deleted. Bottom, representative northern blot showing a dose-
dependent response of PAN-WT and PAN-D79 to ORF57. Cells were
co-transfected with PAN and ORF57 expression plasmids as indicated.
Because these constructs use the ORF50-dependent PAN promoter, an
ORF50 expression plasmid was also co-transfected. Control panels show
signal from a co-transfected plasmid that controls for transfection and
loading efficiencies. (B) Quantitation of dose-dependent experiments
shown in (A). Values are normalized to the no ORF57 control lanes; error
bars show standard deviation (n=4).
KSHV ORF57 Binds and Stabilizes PAN RNA
PLoS Pathogens | www.plospathogens.org3 March 2010 | Volume 6 | Issue 3 | e1000799
accumulation differences are due to this difference. All transcripts
utilize the PAN RNA polyadenylation signal.
Found at: doi:10.1371/journal.ppat.1000799.s003 (0.29 MB TIF)
ORF57. TRP-D79 was transfected into 293A-TOA cells and the
transfected cells were used for in situ hybridization with PAN
RNA probes (middle). PAN RNA signal is shown in the presence
and absence (vector) of ORF57 as indicated. Nuclei are stained
with DAPI (left) and merged images are shown (right panels).
Found at: doi:10.1371/journal.ppat.1000799.s004 (6.40 MB TIF)
TRP-D79 RNA remains nuclear in the presence of
We thank Dr. Jens Lykke-Andersen for plasmids used in this study, Drs.
Julie Pfeiffer and Pinghui Feng for critical review of this manuscript, Dr.
Neal Alto for help with confocal microscopy, and Olga Volovnik for
Conceived and designed the experiments: NKC. Performed the experi-
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PLoS Pathogens | www.plospathogens.org15 March 2010 | Volume 6 | Issue 3 | e1000799