Download Alternative splicing in human tumour viruses

Biochem. J. (2012) 445, 145–156 (Printed in Great Britain)
145
doi:10.1042/BJ20120413
REVIEW ARTICLE
Alternative splicing in human tumour viruses: a therapeutic target?
Hegel R. HERNANDEZ-LOPEZ and Sheila V. GRAHAM1
Centre for Virus Research, Institute of Infection Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, Scotland, U.K.
Persistent infection with cancer risk-related viruses leads to
molecular, cellular and immune response changes in host
organisms that in some cases direct cellular transformation.
Alternative splicing is a conserved cellular process that
increases the coding complexity of genomes at the pre-mRNA
processing stage. Human and other animal tumour viruses use
alternative splicing as a process to maximize their transcriptomes
and proteomes. Medical therapeutics to clear persistent viral
infections are still limited. However, specific lessons learned in
some viruses [e.g. HIV and HCV (hepatitis C virus)] suggest
that drug-directed inhibition of alternative splicing could be
useful for this purpose. The present review describes the basic
mechanisms of constitutive and alternative splicing in a cellular
context and known splicing patterns and the mechanisms by
which these might be achieved for the major human infective
tumour viruses. The roles of splicing-related proteins expressed
by these viruses in cellular and viral gene regulation are explored.
Moreover, we discuss some currently available drugs targeting
SR (serine/arginine-rich) proteins that are the main regulators of
constitutive and alternative splicing, and their potential use in
treatment for so-called persistent viral infections.
INTRODUCTION
pathobiology of cancer caused by tumour virus–host cell
interaction is highly complex, but it is known to include viral
oncogene expression at an elevated level. The mechanisms behind
this are myriad and include viral genome integration into the host
genome, host cell epigenetic changes and modification of host cell
protein expression, virus-induced immunosuppression that may
activate other tumour viruses, chronic inflammation, prevention of
apoptosis, induction of chromosomal instability and chromosomal
translocations [2]. A common requirement in virally-induced
cancer development is persistent infection with the virus in
question: long-term infection initiates cellular changes that
predispose to cancer progression. Currently, anti-viral vaccines
are mainly developed to prevent primary infection and usually
do not have any role in treating chronic or persistent infection
[10]. A better understanding of virus–host cell interaction and
viral life cycles has recently led to refocusing of research into
preventing persistent infection, particularly development of drugs
targeting cellular factors that are essential for persistent viral
infection. This approach may be advantageous because it would
reduce the risk of the emergence of viral drug resistance, a major
risk if virus proteins were targeted [11]. There are a number of
possible cellular targets for antiviral therapy, for example, viral
entry via cellular receptors [12], microRNAs [13], mitochondrial
metabolism [14], innate immunity [15], cellular sites of virus
assembly [16] and virus exit from the cell [17], nuclear transport
[18], viral RNA processing [19] and viral protein translation [20].
Up to 20 % of human cancers are causally related with infectious
diseases. Of these, the majority have a viral aetiology [1,2]. RSV
(Rous sarcoma virus), a retrovirus that induces avian sarcomas,
was the first tumour-related virus recognized [3,4] and launched
the study of viral infections as a cause of cancer. CRPV (Cottontail
rabbit papillomavirus) was the first mammalian tumour-inducing
DNA virus described [5], and identification of the association of
several other mammalian tumour viruses with cancer progression
has since followed [6]. The association of human cancer and
tumour viruses was confirmed with the discovery of EBV
(Epstein–Barr virus), the primary cause of B-cell transformation
in Burkitt’s lymphoma [7]. Currently, the International Agency
for Research on Cancer (http://www.iarc.fr/; [8]) classifies six
human viruses as ‘carcinogenic to humans’: two herpesviruses,
HHV-4 (human herpesvirus 4), also known as EBV, and HHV8, also known as KSHV [KS (Kaposi’s sarcoma)-associated
herpesvirus]; two liver-infecting hepatitis viruses, HBV (hepatitis
B virus) and HCV (hepatitis C virus); one human retrovirus,
HTLV-1 (human T-cell leukaemia virus type 1); and 15 genotypes
of the so-called ‘high risk’ HPVs (human papillomaviruses).
Lastly, a newly discovered tumour virus, MCPyV (Merkel cell
polyomavirus) was found through deep sequencing of Merkel
cell tumour DNA [9]. All of these viruses commonly infect
the human population, yet rarely cause serious disease. The
Key words: alternative splicing, cancer, disease therapy, tumour
virus.
Abbreviations used: AON, antisense oligonucleotide; BBP, branch-point-binding protein; CLK, cdc2-like kinase; DMD, Duchenne muscular dystrophy;
DYRK, dual-specificity tyrosine-phosphorylated and -regulated kinase; EBNA, EBV nuclear antigen; EBV, Epstein–Barr virus; ESE, exonic sequence
enhancer; ESS, exonic sequence silencer; HBV, hepatitis B virus; HBSP, HBV splice-generated protein; HCV, hepatitis C virus; HHV, human herpesvirus;
hnRNP, heterogenous nuclear ribonucleoprotein; HPV, human papillomavirus; HTLV, human T-cell leukaemia virus; IDC, indole derivatives compound; IL,
interleukin; KS, Kaposi’s sarcoma; KSHV, Kaposi’s sarcoma-associated herpesvirus; LANA, latency-associated nuclear antigen; LMP, latent membrane
protein; MAPK, mitogen-activated protein kinase; PS, polymerase-surface; RexRE, Rex-response element; RS, arginine/serine; SA, splice acceptor; SD,
splice donor; shRNA, small hairpin RNA; siRNA, small interfering RNA; SMN, survival of motor neuron; snRNP, small nuclear ribonucleoprotein; SR,
serine/arginine-rich; SRPK, SR protein kinase; SRSF, SR-splicing factor; ss, splice site; STAT, signal transducer and activator of transcription; TOES,
targeted oligonucleotide enhancers of splicing; U2AF, U2-associated factor; vCCL, virally-encoded chemokine; vIRF, viral interferon-response factor;
vGPCR, viral G-protein-coupled receptor.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 1
H. R. Hernandez-Lopez and S. V. Graham
The basic splicing machinery
Key cis -acting sequences (upper-case letters) and the proteins or protein–RNA complexes
(snRNPs) that they bind at exon/intron junctions (5 -/3 -ss, downward arrows) or internal to
introns are shown. Exons are shown as pink boxes. The intron is shown as a black line. snRNPs
are shown as large shaded ovals. The U2AF dimer is shown in green-shaded beige. BBP is
shown as a purple shaded oval. Blue arrows indicate interactions.
In particular, examination of the mechanisms of regulation of the
latter three processes is revealing well-founded novel therapeutic
targets against chronic infections by tumour viruses.
CONSTITUTIVE AND ALTERNATIVE SPLICING
Constitutive splicing consists of the removal of all pre-mRNA
introns (non-coding region) and the splicing together of every
exon (coding region) of a gene to obtain a mature mRNA.
The discovery of splicing in adenovirus-2 [21,22] triggered
the accumulation of new knowledge not only in the regulation
of eukaryotic gene expression, but also regarding regulation of
the highly complex cellular protein machinery called the
‘spliceosome’ that is required to accomplish pre-mRNA splicing,
a highly ordered process. The spliceosome machinery consists
of five core snRNPs (small nuclear ribonucleoproteins), U1,
U2, U5 and U4/U6, and up to 300 other proteins involved in
splicing [23]. Pre-mRNA recognition by snRNPs starts with
recognition/interaction of BBP (branch-point-binding protein)
with the intron branch point and binding of U2AF (U2-associated
factor; 65 and 35 kDa subunits) to the polypyrimidine tract
within the intron, upstream of the 3 -ss (3 -splice site) or
Figure 2
SA (splice acceptor) site. Recruitment of U1 snRNP to the 5 -ss
or SD (splice donor) site follows. Next, recruitment of U2 snRNP
to the branch point is achieved through interaction with U2AF.
The U1–pre-mRNA–U2 complex interacts with the tri-snRNP
complex, U4–U5–U6 snRNPs and conformational rearrangement
of the resulting snRNP complex leads to the first SN 2-type
transesterification reaction, which generates a free 5 -exon and an
intron lariat joined to the 3 -exon. Finally, the 3 -hydroxy group
of the 5 -exon attacks the 3 -ss at the phosphodiester bond by a
second transesterification reaction, leading to exon–exon ligation
and release of the lariat intron [24,25]. The key cis-acting signal
sequences and the proteins/complexes they recruit are shown in
Figure 1.
Once formed on pre-mRNA substrates, the snRNP complexes
are stabilized and regulated by two main classes of proteins, the SR
(serine/arginine-rich) protein family and hnRNPs (heterogeneous
nuclear ribonucleoproteins). SR proteins generally enhance
splicing reactions by binding to ESEs (exonic sequence
enhancers) or ISEs (intronic sequence enhancers) and interact
with the basic splicing machinery to recruit snRNPs to SD or
SA sites [26]. These proteins also help define exons and introns
in splicing by creating connections between snRNPs along the
length of the pre-mRNA (Figure 2). There are nine classical
SR proteins named SRSF (SR-splicing factor) 1–9, and are
highly conserved in all metazoans and plants [26]. Several other
important non-classical SR proteins, such as Tra2β (SRSF10),
play important regulatory roles in development and differentiation
[27,28]. All SR proteins contain a SR domain and at least
one RRM (RNA-recognition motif). hnRNPs are RNA-binding
proteins that generally display an antagonistic function to SR
proteins in ss selection [29]. hnRNP proteins bind ESS (exonic
sequence silencers) or ISS (intronic sequence silencers) in a cooperative fashion to interfere with SR protein binding (Figure 2)
[30].
Alternative splicing is the key way in which a single gene can
produce more than one protein product. For example, the human
genome contains approximately 23 000 genes, yet expresses over
90 000 proteins. It has been recognized that up to 95 % of human
genes can give rise to alternatively spliced mRNAs in different
human tissues. Alternative splicing is an essential process in
development, differentiation and normal cell function [26]. There
are a number of distinct mechanisms by which different mRNA
isoforms may be generated: exon skipping, intron retention,
Splicing factor interactions on a pre-mRNA
Diagram of possible regulatory interactions across exons and introns (double-ended arrows), and between the cap at the 5 -end and the cleavage and polyadenylation machinery at the 3 -end, within
a single putative mRNA. Positive ( + ) stimulatory activities of SR proteins (blue shaded ovals) and negative ( − ) antagonistic activities of hnRNP proteins (hn; green shaded ovals) are indicated.
The ESE is shown as a light orange box and the ESS as a green box. The 5 -ss is the SD site and the 3 -ss is the SA site. U1 snRNP is shown as a pink shaded oval (U1), U2 snRNP is shown as a
yellow shaded oval (U2) and U2AF is shown as a beige shaded oval. Exons are shown as blue shaded boxes, and introns and 5 - and 3 -untranslated regions are shown as black lines. The mRNA
cap is shown as an olive oval. The cleavage and polyadenylation machinery is shown as fuschia ovals and the polyadenylation site [poly(A)] is indicated with a downward arrow.
c The Authors Journal compilation c 2012 Biochemical Society
Viruses, drugs and alternative splicing
Figure 3
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Possible products of alternative splicing of a hypothetical gene
(A) Structure of a three-exon, two-intron gene. Exons are illustrated in dark blue and introns in light blue. Two alternative promoters, one upstream of the coding region (Pwt ) and one in the first
exon (Palt ), are shown as black arrows. Two alternative polyadenylation sites are shown as blue downward arrows and indicated as poly(A)wt for the most frequently used polyadenylation site and
poly(A)alt for an alternative polyadenylation site in intron 2. (B) Some examples of possible mRNAs that could arise from transcription using alternative promoters and using alternative splicing and
polyadenylation. The 3 -untranslated region is shown as a black line.
alternative ss selection, alternative promoter usage and alternative
polyadenylation, with the former three being controlled by the
splicing machinery (Figure 3).
As well as controlling constitutive splicing, SR proteins
regulate alternative splicing by promotion of proximal 5 -ss
selection. Similarly, hnRNP proteins contribute to alternative
splicing regulation by repression of ‘cassette’ exon inclusion
in certain mRNAs [29]. Several diseases (e.g. spinal muscular
atrophy, retinitis pigmentosa, tauopathies, thalassaemias and
familial dysautonomia) have been related with alternative
missplicing [31,32]. Alternative splicing defects also underlie
cancer formation and development [33] and SR proteins 1 and
3 have been shown to have roles in oncogenic transformation
[34,35] by regulating production of oncogenic splice isoforms of
key cellular proteins. For example, SRSF1 regulates production
of anti-apoptotic isoforms of the tumour suppressor BIN1
(bridging integrator 1) and stimulates synthesis of specific
oncogenic isoforms of the kinases Mnk2 [MAPK (mitogenactivated protein kinase)-interacting serine/threonine kinase] and
S6K1 (S6 kinase) that circumvent normal translation regulation
by eIF4E (eukaryotic initiation factor 4E) phosphorylation via
the Ras/MAPK and PI3K (phosphoinositide 3-kinase)/mTOR
(mammalian target of rapamycin) signalling pathways [34].
The function of SR phosphoproteins in constitutive and
alternative splicing is regulated by cycles of phosphorylation/dephosphorylation of the RS (arginine/serine) domain of
the proteins. Accurate control of SR protein phosphorylation
is the main regulatory mechanism for assembly of the splicing
machinery and control of the splicing reaction. Importantly,
phosphorylation also regulates the nuclear/cytoplasmic shuttling
of most SR proteins. The leading protein kinase families that
orchestrate this modification include the SRPK (SR protein
kinase) family [36,37], CLK (cdc2-like kinase) family [38] and
topoisomerase 1 [39]. Members of more than one of these kinase
families may co-operate to regulate a single SR protein. For
example, the prototypical SR protein SRSF1 is phosphorylated
by both SRPK1 and CLK1 proteins [40] to regulate its cellular
localization and splicing activity.
ALTERNATIVE SPLICING IN HUMAN TUMOUR VIRUSES
Economy in genome size is a common theme of most viruses.
To achieve expression of the maximum number of proteins from
small genomes, viruses utilize multiple promoters, overlapping
open reading frames, antisense transcription, translational frame
shifting and stop codon read-through. Notably, alternative splicing
is a key mechanism used by most DNA viruses and nuclear
replicating RNA viruses to generate the full repertoire of viral
proteins. This strategy is used during the normal infectious life
cycle of these viruses and is required for efficient viral replication.
On the other hand, cellular pre-mRNA splicing is a target of
a number of important viruses, such as herpes simplex virus
and influenza virus, which produce viral proteins that inhibit
cellular splicing. Splicing regulation is clearly also important in
tumorigenesis caused by tumour viruses. In some cases, viral
oncogene transcripts are required to be alternatively spliced
to produce tumour-promoting protein isoforms. Moreover, the
growth-promoting properties of tumour viruses can lead to cellular
alterations that promote expression of splicing factors. In this case,
as mentioned above, increased expression of splicing regulators
can lead to production in the cell of novel or altered mRNA
isoforms whose protein products have tumour-promoting activity.
In the present review the possible mechanisms operating in each
human tumour virus will be discussed.
EBV (HHV-4)
EBV is a double-stranded DNA virus that preferentially infects
human B-lymphocytes. It is present in over 90 % of the human
population following infection during childhood. Normally, no
disease occurs, but if primary infection with the virus is delayed
until adolescence, infectious mononucleosis ensues. However, in
certain circumstances, long-term latent infection with EBV in
lymphocytes is associated with lymphoproliferative diseases and
at least three human tumours, Burkitt’s lymphoma, nasopharygeal
carcinoma and Hodgkin lymphoma (Table 1). As with other
herpesviruses, EBV latent infection is characterized by a low
c The Authors Journal compilation c 2012 Biochemical Society
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Table 1
H. R. Hernandez-Lopez and S. V. Graham
Tumour viruses: genes, proteins and associated malignancies
v, viral; ?, unknown.
Tumour virus Oncogenes
Other genes involved in
oncogenesis
Viral alternative spliced
genes/products
Cellular controlled AS
genes
AS proteins involved
Associated malignancies
Burkitt’s lymphoma, Hodgkin
lymphoma, post-transplant
lymphoma, X-linked
lymphoproliferative
syndrome, T-cell
lymphomas,
nasopharyngeal carcinoma
Kaposi’s sarcoma, primary
effusion lymphomas,
multicentric Castleman’s
disease
Hepatocellular carcinoma
Hepatocellular carcinoma
Cervical carcinoma, vaginal
carcinoma, anal carcinoma,
penile cancer, squamous
cell carcinoma, actinic
keratosis, head and neck
squamous cell carcinomas
Adult T-cell leukaemia
EBV
LMP1, EBNA2
EBNA1, EBNA-LP , EBNA3A ,
EBNA3C , BARF1
BLLF1/BLLF , EBNA1, EBNA2 , STAT1, P450arom *,
EBNA3A , EBNA3B ,
CD44 *
EBNA3C , EBNA-LP ,
BZLF1, A73
SM, SRSF3, SRSF10*,
hnRNPs*
KSHV/HHV8
LANA , vCyclin , vFLIP ,
Kaposins , K1, K15 , vIRFs ,
vCCLs , vGPCR , vIL-6 ,
vBCL-2 , RTA , K-bZIP
HBx
Kaposins , vCyclin.90 , vIAP
Kaposins (ORF K12) , RTA ?,
K15 , ORF73/ORF72/
ORFK13 , ORF50/ORFK8/
ORFK8.1, ORF 59
HBSP, PS
?
ORF57, SRSF1, SRSF4,
SRSF6, SRSF5,
U2AF, hnRNP K
?
?
SRSF1, SRSF2,
SRSF3
SR*, SRPK*
SRSF1, SRSF2, SRSF3,
hnRNP A1
?
?
HBV
HCV
HPV
HTLV
?
E6 , E7
E6 , E7 , E1, E4 , L1
Tax
X-region , env , rex , tax,
mRNA: rof , tof
*Unknown mechanism.
rate of virus replication and expression of a limited set of viral
proteins, including the known EBV proteins with oncogenic
activity, EBNA (EBV nuclear antigen) 2, EBNA3A and EBNA3C,
LMP1 (latent membrane protein 1) and BARF1 (BamHI-A
reading frame-1). EBNA2 and LMP1 are absolutely required
for cellular transformation. EBNA2 activates transcription of
viral and cellular genes, including LMP1, LMP2 and the c-myc
oncogene. Other latent proteins, such as EBNA1 and EBNA-LP,
may play an important role in this process (Table 1) [41–44].
Most of these proteins are the products of the activity of different
promoters (Cp, Wp, Qp) that generate long pre-mRNAs that are
alternative spliced and polyadenylated [42,45,46]. To date, the
cellular regulatory proteins implicated in EBV alternative splicing
regulation during latency have not been elucidated.
In the case of lytic replication, expression and phosphorylation
by protein kinase CK2 [47] of the viral SM protein (also called
EB2, Mta and BMLF1) is essential. SM protein is an RNAbinding phosphoprotein that regulates viral [48–52] and cellular
mRNA expression [53,54]. In a microarray-based analysis of EBV
gene expression, SM expression was shown to be necessary for
viral DNA replication and for the expression of approximately
50 % of EBV genes, including the viral oncogenes EBNA2 and
LMP1 [50]. The mechanism of post-transcriptional regulation by
SM is mainly through binding target mRNAs and includes control
of mRNA stability, mRNA nuclear export and alternative splicing
[51–61]. SM protein promotes accumulation in the cytoplasm of
a specific viral unspliced BLLF1 mRNA that encodes gp350, a
structural protein, and other mRNAs (BcLF1, BDLF1, BFRF3,
BdRF1/BVRF2 and BVRF1) encoding EBV capsid proteins. So
SM protein is absolutely necessary for viral capsid formation
and DNA encapsidation, and therefore for production of EBV
infectious virus [49]. Recently, using deep sequencing of EBVpositive Burkitt’s lymphoma cell RNAs, further evidence was
found for novel viral splice isoform mRNA production, but the
impact of this has yet to be worked out [45,62]. Important new
data have revealed that SM protein can affect cellular alternative
c The Authors Journal compilation c 2012 Biochemical Society
splicing. It has been shown to increase expression of both isoforms
of the cellular interferon regulatory transcription factor STAT1
(signal transducer and activator of transcription 1), STAT1α and
STAT1β, but with a disproportionately higher amount of STAT1β.
Increased expression of STAT1β is accompanied by use of an
alternative 5 -ss. The alternatively spliced mRNA is translated to
give a protein lacking the terminal 38 amino acids. This truncated
protein can have dominant-negative effects on transcriptional
regulation of interferon production. Reduced interferon response
would provide a cellular environment conducive to EBV
replication and may lead to persistent infection that underpins
cancer formation. The regulation of STAT1 alternative splicing
by SM is RNA sequence-dependent and can be antagonized by
SRSF1 [53]. Indeed, the pattern of binding of SM to viral mRNAs
indicates that it possesses hnRNP-like activity [51]. SM can also
bind SRSF3, and down-regulation of this SR protein by siRNA
(small interfering RNA) reduced the alternative splicing of STAT1
RNA significantly [54]. SM has also been found to interact with
Tra2β (SRSF10) [63] and to be present in cellular complexes that
include hnRNP A1/A2 and hnRNP C1/C2 [55]. In EBV gene
regulation, SM may mediate alternative splicing by recruiting
spliceosomes on to viral pre-mRNAs, in a similar manner to
SR proteins, in order to stimulate efficient lytic replication
[54]. Apart from the SM protein, viral EBNA-LP and EBNA1 proteins associate with SRSF1 [64,65] and other splicingrelated proteins, such as SF3B (splicing factor 3B subunit 2), U1A
(U1 small nuclear ribonucleoprotein A), U2B (U2 small nuclear
ribonucleoprotein B) and several members of the hnRNP family
(e.g -A/B, -A1, -K, -M, -K, -R and -U) were identified by MS
in a co-immunoprecipitation with EBNA-LP [64]. The functional
consequence of these interactions has yet to be explored.
KSHV (HHV-8)
KSHV is the second herpesvirus known to be associated with
cancer. Although the main cell types that are infected in vivo
Viruses, drugs and alternative splicing
remain controversial, KSHV is fully associated with three
lymphoproliferative conditions: KS, PEL (primary effusion
lymphoma) and multicentric Castleman’s disease (Table 1).
KSHV-induced oncogenesis occurs upon reactivation from
latency of the lytic replication cycle of the virus in immunosuppressed individuals, usually in association with HIV/AIDS or
drug-induced immunosuppression. The profile of gene expression
during latent infection includes LANA-1 (latency-associated
nuclear antigen 1), a viral cyclin, a viral FLIP homologue,
virally-encoded microRNAs and kaposins, all encoded in the
latent gene cluster. Meanwhile, the main lytically expressed
genes are the vGPCR (viral G-protein-coupled receptor) K1, the
vIRFs (viral interferon-response factors), viral IL (interleukin)-6
homologue, vCCLs (virally-encoded chemokines) and a multimembrane-spanning protein K15, of which most are expressed in
KS. Although the expression of only latent genes is insufficient
to transform cells, both latent and lytic KSHV gene products
contribute to oncogenesis in an additive way (Table 1) by distinct
mechanisms, such as altered angiogenesis, immune modulation,
altered signal transduction and promotion of cellular growth
and survival [66–68]. Many KSHV genes are encoded in gene
clusters regulated by a common promoter or are co-terminal with
a single polyadenylation signal, leading to expression of some
polycistronic pre-mRNAs which are constitutively and alternative
spliced [69]. KSHV alternative splicing is largely promoted by
the RNA-binding protein ORF57 [70], a homologue of both EBV
SM protein and the paradigm herpes simplex virus type 1 RNA
regulatory protein ICP27 [71], which is phosphorylated in vivo
by CK2 [72]. ORF57 regulates transcription, RNA processing,
nuclear export and RNA stability [70,73–76]. ORF57 expression
is important for viral gene expression and for infectious virion
production [74,77]. It can perform the role of a splicing factor in
spliceosome-mediated viral RNA splicing in vitro, for example,
β-globin splice isoform production, and in vivo in ORFs50/8/8.1
alternative splicing regulation [73]. ORF57 co-localizes with SR
protein SRSF2 in typical nuclear splicing speckles. Moreover, it
is found in complex with cellular snRNAs (small nuclear RNAs)
and splicing factors such as U2AF and hnRNP K [73,78]. The
interaction of ORF57 with elements of the splicing machinery
may indicate the role of this protein in directing ss selection
and recruitment of alternative splicing proteins for pre-mRNA
processing.
HPVs
The Papillomaviridae family contains at least 189 genotypes of
papillomaviruses, of which 120 infect humans [79]. HPV16 and
HPV18 are the most prevalent worldwide and persistent infection
with these types cause approximately 54.4 % and 16.5 % of all
invasive cervical cancers worldwide respectively. The remainder
of cases of cervical cancer is caused by 13 other less prevalent
‘high-risk’ HPV types. HPV infection is also associated with
other anogenital and head and neck cancers and, in co-operation
with UV light, with squamous cell carcinoma and its premalignant disease, actinic keratosis [2]. The molecular hallmarks
of latency and persistence in HPV infection are not yet understood.
Although HPV expresses only approximately eight proteins,
regulation of gene expression, and the viral life cycle in general,
is controlled by cellular and viral factors in response to epithelial
differentiation [80]. The tight linkage between viral replication
and the differentiating epithelium has meant a lack, until recently,
of systems for generation of infectious virus particles and slow
progress in the understanding of key events in the infectious life
cycle. Moreover, it appears that the viral life cycle and viral
149
gene expression and its regulation is rather complex. What is
known is that viral proteins are translated from polycistronic
mRNAs that are extensively alternatively spliced and alternatively
polyadenylated [19].
Oncogenicity of HPV16 is largely attributable to the E6 and
E7 oncoproteins [81]. In a normal infection, only low levels
of these proteins are required to stimulate the cell cycle of
differentiated, non-dividing keratinocytes in order that a cellular
environment that allows viral genome synthesis is achieved [82].
So E6 abrogates apoptosis/differentiation and E7 stimulates cell
cycle G1 to S-phase transition in differentiated cells by interacting
with p53 and pRb (retinoblastoma protein) respectively. In a
persistent infection, E6/E7 expression is increased either through
integration of the viral genome, which counteracts the normal
virally mediated repression of oncoprotein expression, or through
epigenetic or other, as yet undefined, changes [83]. E6 and E7
are expressed from polycistronic alternatively spliced isoforms
in most of the ‘high-risk’ HPV types (Figure 4B). One of the
isoforms probably encodes E6 (full-length E6), whereas the other
encodes E7 (E6*I) [84]. HPV16 has a particularly complex E6/E7
alternative splicing pattern with at least four different isoforms
expressed (Figure 4C). Therefore HPV oncoprotein expression
relies on alternative splicing [85,86]. The mechanisms controlling
this are not fully elucidated, although SRSF3 has been implicated,
because, upon its knockdown, levels of the oncoproteins were
significantly reduced [87]. Two of the proteins encoded by the
HPV16 genome have been proposed to have splicing-regulatory
functions. The viral oncoprotein E6 and the viral E2 replication
and transcription factor have both been shown to inhibit splicing of
reporter constructs in vitro. Moreover, these two proteins possess
RNA-binding activity and can interact with SR proteins [88]. The
results from that study stand in contrast with an earlier study by
Lai et al. [89] where the skin-infective HPV5 E2 protein, which
possesses an RS-rich hinge region, could bind SR proteins and
two snRNP proteins; this interaction was not found in the first
study. Moreover, Lai et al. [89] found that HPV5 E2 was able
to stimulate splicing of an intron-containing reporter gene in an
in vitro system, but other HPV E2 proteins, including that of
HPV16, which all possess shorter RS domains, failed to bind
SR proteins. Further investigation is required to elucidate these
contradictions.
Investigation of cis-acting sequence elements that might bind
SR or hnRNP proteins in the HPV genome has revealed an ESE
in the middle of the HPV16 E4 open reading frame. Investigation
of its function in an HPV subgenomic construct showed that
it controls the adequate and efficient recognition of a weak
3 -SD site downstream and regulates the switch from the use
of the early polyadenylation site at the end of the early gene
region to the late polyadenylation site at the end of the late
gene region (Figure 4A) [90]. A very similar element exists in
bovine papillomavirus-1 and binds SRSF3 in a concentrationdependent manner [87]. SRSF1 has also been shown to bind
within the same HPV16 open reading frame, but at a different
region just downstream of the viral major early SA site and to
regulate selection of the same weak 3 -ss [91]. Upon mutational
inactivation of the SRSF1 ESE, or introduction of a mutant SRSF1
protein unable to bind this element, splicing was redirected to
another 3 -ss at the beginning of the L1 open reading frame.
SRp30c (SRSF9) and PTB (polypyrimidine tract binding protein;
hnRNP I) have also been shown to regulate splicing from HPV
subgenomic constructs [92]. Moreover, a subset of SR proteins,
SRSF1, SRSF2 and SRSF3, are up-regulated by HPV during
infection in response to epithelial differentiation. The viral E2
transcription factor controls expression of all three, probably
through transcriptional trans-activation of their promoter, as
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150
Figure 4
H. R. Hernandez-Lopez and S. V. Graham
Alternative splicing of the HPV16 E6 and E7 pre-mRNAs
(A) Diagram of the HPV16 genome showing the eight open reading frames (open boxes annotated with the gene name E or L above or below the box), the early and late polyadenylation sites
[poly(A)] and the main SD (orange arrows) and SA (blue arrows) sites. (B) Three selected mRNAs encoding the HPV16 E6 and E7 oncoproteins to show that they are expressed as part of
polycistronic mRNAs. For ease of illustration, only one splicing event is shown in the E6 and E7 open reading frames. (C) Several alternative splicing events can occur to produce the HPV16
E6/E7-encoding mRNAs. At the top is shown the E6/E7 region of HPV16 genome. P97 indicates the major genome promoter. Other promoters exist but are not illustrated in this Figure. Below are the
structures of four RNA splice isoforms encoding the viral oncoproteins. Splice junctions are numbered according to their genomic location in nucleotides. SD sites are indicated with orange arrows
and the alternative SA sites are indicated with blue arrows.
demonstrated for SRSF1 [93]. This regulation is specific for the
three smallest SR proteins because levels of other SR splicing
factors of greater molecular mass are not altered during HPV
infection or upon overexpression of HPV E2 protein [94].
Moreover, the same three SR proteins are overexpressed during
cervical tumour progression, indicating that they may contribute
to progression of transient to persistent viral infection underlying
tumour formation [94]. SRSF1 phosphorylation has also been
shown to be altered in HPV infection [94,95] in an epithelial
differentiation-dependent manner, suggesting alterations in SR
protein function during the HPV replication cycle. SRPK1, which
can phosphorylate SRSF proteins, physically associates with
the viral E4 protein of HPV1, HPV16 and HPV18, suggesting the
possibility of manipulation of the alternative splicing cellular
machinery by these viruses [96]. Intriguingly, HPV E2 and E4
proteins can be found together in infected cell protein complexes,
so combinatorial control of splicing by these two proteins during
virus infection may be possible [97].
HBV and HCV
Belonging to the Hepadnaviridae and Flaviviridae families
respectively, HBV and HCV are responsible for acute and
chronic hepatitis, besides being the major aetiological agents
of HCC (hepatocellular carcinoma). Both are enveloped noncytopathic hepatotropic viruses. HBV is a partially doublestranded DNA virus that replicates via an RNA intermediate,
whereas HCV contains a positive sense single-stranded RNA
genome. Robust culture systems for HCV propagation were
developed only recently [98], so many aspects of the HCV life
c The Authors Journal compilation c 2012 Biochemical Society
cycle and gene expression patterns remain unclear. There are
common well accepted mechanisms of cancer development by
both viruses. Among these are changes in micro-environment,
chronic necroinflammation, hepatocyte necrosis/regeneration
rate, deregulation of signalling pathways and oxidative stress. For
HBV, the HBx protein encoded by the X gene has been proposed
to have oncogenic functions, but the precise mechanisms by
which HBx causes tumour progression remain unclear. However,
HBx trans-activates several gene pathways, including p53, Notch
and Bcl-2 signalling, that could be involved in the genesis of
hepatocarcinoma [99,100]. HBV produces three large unspliced
RNAs that can be spliced to produce mRNAs encoding viral
structural and non-structural proteins. To date, five splice-donor
and five splice-acceptor sites have been identified [101]. Although
the possible combinations can be numerous, the proteins reported
thus far that are expressed from these alternatively spliced
RNAs are HBSP (HBV splice-generated protein) [102] and
the endoglycosidase H-sensitive PS (polymerase-surface) fusion
protein [103]. Although the HBV-spliced transcripts appear to
be dispensable for viral replication, PS splice variant expression
may control the virus life cycle in some way, because HBV
DNA replication was reduced in the presence of PS spliced
transcript and protein [104]. Moreover, viral persistence and
severity of hepatic disease may depend on HBSP splice variant
production [102,105]. SRPK1 and SRPK2, two important kinases
involved in cycles of SR activation/inactivation, are able to
phosphorylate HBV core protein in vitro, a step necessary for
viral genome encapsidation [106]. In contrast, both SRPK proteins
were found to suppress HBV DNA replication at the pre-genome
packaging stage, an event that did not rely on their kinase activities
[107].
Viruses, drugs and alternative splicing
Figure 5
151
Splicing of the HTLV pX region and its regulation
(A) Diagram of the HTLV-1 genome showing the open reading frames (open boxes with associated gene names). (B) The mRNAs encoding pX transcribed from the HTLV genome. ex1, exon 1. ex2,
exon 2. Heavy continuous lines indicate coding regions, light continuous lines indicate intron sequences that are spliced out. Numbers indicate the genomic positions in nucleotides of the SA sites
in the pX gene region. The regions associated with hnRNP A1 (hnA: green oval) and SRSF1 (SR1: blue oval) binding to control alternative splicing are indicated with arrows.
HCV mRNA does not undergo splicing. However, studies
have demonstrated that the cellular RNA helicase DDX3, which
is regulated by HCV infection, is actually required for HCV
replication [108,109]. DDX3 has multiple known roles in
RNA metabolism, including splicing [110]. DDX3 can interact
with the yeast spliceosome and human mRNPs (messenger
ribonucleoproteins) [111,112] and possesses an RS domain-like
region in its C-terminus, suggesting that it might bind or function
as an SR protein [113]. Arguing against a role in splicing itself
is the finding that DDX3 binds fully-formed mRNAs and not
pre-mRNAs [111]. DDX3 has roles in innate immunity to viruses
and may also regulate translation, cell cycle and apoptosis [114],
meaning that its ability to regulate HCV replication (and the
replication of other medically important viruses, such as HIV
and poxviruses) may not be exerted through RNA metabolism.
HTLV
HTLV type 1 causes adult T-cell leukaemia/lymphoma in
populations in Japan, the Caribbean, South America and Africa.
HTLV is a retrovirus that expresses one major oncoprotein, the
Tax transcription factor. Tax binds TRE1 (Tax-responsive element
1) in the viral LTRs (long terminal repeats) and trans-activates
its promoter [115]. Tax can also trans-activate transcription
of cellular genes via recruitment of NF-κB (nuclear factor
κB), CREB (cAMP-response-element-binding protein) and SRF
(serum response factor). Tax has roles in initiating oncogenic
transformation, but, unlike the oncoproteins of other tumour
viruses, it appears that continued expression of Tax is not required
for maintenance of the tumour phenotype. Tax is expressed from
the pX gene region at the 3 -end of the HTLV genome. This
region contains at least five open reading frames, and mRNAs
are generated by alternative splicing, including or excluding
exon 2 and utilization of alternative 3 -ss (Figure 5). One of
these alternatively spliced mRNAs encodes the RNA-binding
protein Rex that is the key regulator of HTLV RNA splicing
and export of intronless (encoding gag-pol-pro) and single intron
(encoding env) transcripts [115]. Rex binds a RexRE (Rexresponse element) within viral RNAs via an N-terminal RNAbinding domain. However, this interaction has not been shown
to be important for any function of Rex in splicing regulation.
Early results suggested that Rex was able to control the ratio of
unspliced to spliced HTLV RNAs [116]. Subsequently, Rex has
been shown to bind hnRNP A1 and its antagonistic SR protein,
SRSF1. hnRNP A1 can compete with Rex for binding to the
RexRE [117] and regulates the relative levels of unspliced and
spliced viral transcripts [118]. siRNA knockdown of Rex resulted
in increased levels of the unspliced gag-pol-pro mRNA, no
change in the spliced env mRNA, but some increase in
tax/rev mRNAs [118]. Another study examined the effect of
overexpression of hnRNP A1 on production of the pX region
transcripts. The results revealed that increased levels of hnRNP A1
promoted exclusion of exon 2, resulting in increased production
of the pX mRNAs containing two, as opposed to three, exons
(Figure 5). In the same study, overexpression of SRSF1 promoted
use of the most distal 3 -ss at nucleotide 6950, but did not alter
exon 2 inclusion [119]. Although it is clear that accurate splicing is
essential for HTLV replication, the exact mechanisms controlling
this are yet to be fully elucidated.
DRUGS THAT CAN MODULATE ALTERNATIVE SPLICING
Extensive chemical screens have been carried out to find drugs
that inhibit spliceosome assembly and splicing, and during the
past few years several candidate drugs have emerged. Research
has focused on modifying incorrect splicing, leading to the
identification and synthesis of a great number of low-molecularmass compounds with this property. For example, clotrimazole,
flunarazine and chlorhexidine, identified in a small-molecule
screening study, were found to have unexpected properties in
controlling alternative splicing. None of these molecules exhibited
a general inhibitory effect on splicing. Instead, each inhibitor had
selective effects on alternative splicing [120]. Examination of
the mechanisms behind this for one of the drugs, chlorheximide,
demonstrated an inhibition of CLKs, one of the families of
kinases that phosphorylate SR proteins. The screening for and
development of other drugs targeting RNA-binding proteins
demonstrates the potential therapeutic use of targeting these
proteins (Table 2). Different types of molecules have been found
to alter alternative splicing and can be grouped into HDAC
c The Authors Journal compilation c 2012 Biochemical Society
152
Table 2
H. R. Hernandez-Lopez and S. V. Graham
Small-molecule names, types and molecular targets for inhibition of alternative splicing
Compound
Drug type
Systematic name
Mechanism
Reference
IDC13
Indol derivative
SR proteins*
[138]
IDC16
IDC78
Indol derivative
Pyridocarbazole
Topo 1, SRSF1
SR proteins*
[131]
[138]
NB-506
Indolocarbazole derivative
Topo 1
[134]
KH-CB19
Leucettine L41
Dichloroindolyl enaminonitrile
Leucettamine B derivative
CLK1, CLK4
CLKs, DYRKs
[136]
[137]
Isodiospyrin
Diospyrin derivative
Topo 1
[132,133]
SRPIN340
Chlorhexidine
Camptothecin
Isonicotinamide derivative
Biguanide
Alkaloid
N -5,6-Dimethyl-5H -pyrido[3 ,4 :4,5]pyrrolo[2,3-g ]isoquinolin-10-yl-N -ethylpropane-1,3-diamine
10-Chloro-2,6-dimethyl-2H -pyrido[3 ,4 ,5]pyrrolo[2,3-g ]isoquinoline
1-Diethylamino-3(9-methoxy-5-methyl-6H -pyrido[4,3-b ]carbazol-1-ylamino)-propan2-ol
6-N -Formylamino-12,13-dihydro-1,11-dihydroxy-13-(β-D-glucopyranosyl)-5H indolo[2,3-a ]pyrrolo-[3,4-c ]carbazole-5,7(6H )-dione
Ethyl-3-[(E )-2-amino-1-cyanoethenyl]-6,7-dichloro-1-methylindole-2-carboxylate
(5Z)-5-(1,3-Benzodioxol-5-yl)methylene-2-phenylamino-3,5-dihydro-4H imidazol-4-one
5-Hydroxy-6-(4-hydroxy-2-methyl-5,8-dioxonaphthalen-1-yl)-7-methylnaphthalene1,4-dione
N -4-[2-Piperidino-5-(trifluoromethyl)phenyl]isonicotinamide
2,2 -(1,6-Hexanediyl)bis(1-{amino[(4-chlorophenyl)amino]methylene}guanidine)
(4S )-4-Ethyl-4-hydroxy-1H -pyrano[3 ,4 :6,7]indolizino[1,2-b ]quinoline3,14(4H ,12H )-dione
SRPK1, SRPK2
CLK2, CLK3, CLK4
Topo 1
[141]
[139]
[140]
*Unknown mechanism.
(histone deacetylase) inhibitors, CLK inhibitors, SRPK inhibitors,
topoisomerase inhibitors, calmodulin kinase inhibitors, MAPK
inhibitors and phosphatase inhibitors [121,122].
Modified nucleic acids, especially AONs (antisense oligonucleotides) and siRNA drugs represent other means of modulating
splicing. Chemical modifications are designed to ensure stability
of the RNAs and increase their binding affinity for the target
sequence in a therapeutic setting. These can include 2 -Omethylation, 2 -fluorylation or 2 -O-methoxylation, LNAs (locked
nucleic acids) or BNAs (bridged nucleic acids). AONs are
designed to work by Watson–Crick base-pairing with the target
RNA. In the case of alternative splicing, the AON acts as a
‘splice-switch’ by binding to exons or exon/intron junctions
to abrogate recognition by the splicing machinery. Perhaps the
best example of this therapeutic approach is modulation of exon
skipping in the dystrophin gene, where mutations induce DMD
(Duchenne muscular dystrophy). The dystrophin gene contains 79
introns and the most common mutation in DMD is exclusion of
exons 48–50, inducing premature translation termination at a stop
codon in exon 51 and production of a non-functional dystrophin
protein [123]. AONs that bind exon 51 have been designed.
These inhibit exon 51 inclusion in the mRNA, thus repairing the
translation defect. Although some exons are still removed in the
final mRNA, the resulting dystrophin protein is at least partially
active. Such AONs are now in clinical trials [124,125]. Similar
approaches are being developed to potentially treat other genetic
diseases, such as dystrophic epidermolysis bullosa [126] and
cancers such as prostate cancer [127]. A related strategy,
called TOES (targeted oligonucleotide enhancers of splicing),
is designed to enhance exon inclusion. SMA (spinal muscular
atrophy) is an autosomal recessive condition caused by mutations
in the SMN1 gene encoding the SMN (survival of motor neuron)
protein. A second copy of the gene, SMN2, exists but cannot
complement the genetic defect in SMN1, because the seventh exon
out of eight in this gene is spliced out in the resulting mRNAs.
A TOES approach has been used to bind to SMN2 exon 7 RNA
and pull in SR proteins to activate inclusion of exon 7 [128].
siRNA or shRNA (small hairpin RNA) approaches have also
undergone extensive testing and show initial promise. si/shRNAs
targeted against an undesired alternatively spliced isoform(s)
of an mRNA encoding a disease-causing protein, if delivered
successfully, should reduce levels of the protein in the affected
cells. Clinical trials using RNA interference against various
c The Authors Journal compilation c 2012 Biochemical Society
diseases and infectious agents, for example shRNA against HIV-1
Tat/rev to inhibit HIV-1 replication, are ongoing [129].
DRUG-INDUCED INHIBITION OF SR PROTEINS
Drug targeting of SR proteins as key positive regulators of
constitutive and alternative splicing is thought to provide a useful
means of modulating splicing. SR inhibitors work by binding
SR proteins directly and altering their affinity for other splicing
factors. In addition, or alternatively, such inhibitors could act
through regulating the kinases (topoisomerase 1, SRPK, CLK)
that control SR protein function. For example, benzopyridoindole
and pyrido-carbazole derivatives [IDCs (indole derivatives
compounds)] can inhibit SR phosphorylation apparently by
binding SRSF1 and/or topoisomerase 1 [130]. The RS domain
present in all SR proteins seems to be the general target for IDCs.
One specific compound, IDC16 (10-chloro-2,6-dimethyl-2Hpyrido[3 ,4 ,5]pyrrolo[2,3-g]isoquinoline) had specific inhibitory
effects on SRSF1. When tested as an anti-HIV drug, it inhibited
HIV-1 splicing and virion production, yet did not alter splicing
of 81 selected cellular RNA with key roles in cell cycle and
apoptosis [131]. The discovery that topoisomerase 1 possesses a
kinase activity towards the RS domain of SR proteins [39] and
could control alternative splicing opened the way to test known
anti-topoisomerase 1 drugs in splicing inhibition. Diospyrin
was found to inhibit spliceosome assembly, whereas diospyrin
derivatives had specific inhibitory effects on different catalytic
steps in splicing [132,133]. Another topoisomerase 1 inhibitor,
a glycosylated indolocarbazole derivative, NB-506, inhibited
phosphorylation of SRSF1, and NB-506-treated HeLa extracts
could not splice in vitro [134]. SRPIN340 {N-[2-(1-piperidinyl)5-(trifluoromethyl)phenyl]isonicotinamide} is a drug developed
to selectively inhibit SRPK1 and SRPK2. It showed no inhibition
of a range of other kinases, including CLK1 and CLK4 [135].
Recently, a novel class of splicing inhibitors (dichloroindolyl
enaminonitriles) was described that are highly specific for
CLK1/CLK4 [136]. Drug inhibition produced a significant change
in the phosphorylation patterns of the classical SR proteins
recognized by the phospho-specific anti-SR protein monoclonal
antibody 104. A CLK and a DYRK (dual-specificity tyrosinephosphorylated and -regulated kinase) inhibitor, leucettine L41,
has been described that inhibits phosphorylation of SRSF4,
Viruses, drugs and alternative splicing
SRSF6 and SRSF7, and modulates alternative splicing in a cellbased reporting system [137]. Other compounds exist that display
wide effects on alternative splicing by different mechanisms. SR
proteins are the main orchestrators of alternative splicing, so their
inhibition shows promise. However, there are many more different
levels of alternative splicing regulation that could be targeted in
the future.
153
FUNDING
H.R.H.-L. is supported through a CONACYT scholarship [number 309211] from the
Mexican Government. The Graham laboratory acknowledges funding from the Wellcome
Trust [grant number 088848/Z/09/Z] and core funding from the MRC-University of Glasgow
Centre for Virus Research.
REFERENCES
SUCCESSFUL HISTORIES IN VIRAL SPLICING INHIBITION
IDCs have been tested in vivo to assess their potential use in
inhibiting SR-dependent alternative splicing of HIV-1 mRNA.
These compounds were capable of repressing the use of several
weak alternative 3 -ss upstream of the open reading frames
encoding the viral proteins Tat, Rev, Vpu, Env and Nef [130].
As mentioned above, one compound, IDC16, showed very
promising capability in the inhibition of virus replication.
IDC16 inhibited production of the key HIV regulatory proteins,
leading to reduced levels of both viral RNA and virus particle
formation [131]. Two further IDCs, IDC13 (N-5,6-dimethyl5H-pyrido[3 ,4 :4,5]pyrrolo[2,3-g]isoquinolin-10-yl)-N -ethylpropane-1,3
diamine)
and
IDC78
{1-diethylamino3(9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazol-1-ylamino)propan-2-ol}, displayed efficient inhibition of early replication
of MLV (murine leukaemia virus) in vivo by altering the
splicing-dependent production of the retroviral envelope protein
in a newborn-infected mouse model [138]. The drugs, when
applied systemically, were effective to the extent of inhibiting
virus-induced pathogenesis. Interestingly, the drugs were welltolerated in the animals and a microarray-based analysis of
cellular splicing revealed little impact of the drug [138]. This
study highlights the need to design drugs that are highly selective
against a particular SR protein implicated in the disease/infection
in question, otherwise there would be deleterious effects on
the cells via major alterations in alternative splicing. A CLK2,
CLK3 and CLK4 inhibitor, chlorhexidine [120], commonly
used in mouthwash solutions, has an inhibitory effect on HIV-1
replication by modifying the alternative splicing pattern of
HIV-1 mRNAs and leading to a decrease in the quantity of the
regulatory viral protein Rev and an indirect reduction in Tat
p14 viral protein [139]. Similarly, it has been suggested that
some topoisomerase 1/2 inhibitor drugs can block KSHV DNA
replication [140]. Testing SRPIN340 on replication of HIV and
Sindbis virus suggested that the compound may have antiviral
properties against acute viruses [135]. SRPIN340 also seems
to be capable of inhibiting the cellular SRPK proteins required
for HCV replication. It is proposed that SRPIN340 has specific
inhibitory effects on HCV subgenomic replication [141].
PERSPECTIVES
The research to date indicates that the development of drugs that
are capable of inhibiting viral alternative splicing shows promise.
Drugs may act either by interfering with the formation of the
cellular splicing machinery, or by targeting the SR activation
pathway via inhibition of SR protein phosphorylation. The
regulation of alternative splicing in tumour viruses seems to
follow all the rules established for cellular splicing. However,
acute viruses may be easier to target than chronic viruses, such as
the majority of tumour viruses. Further directed drug synthesis
and screening for inhibition of splicing in these viruses may
prove challenging. Finally, the emerging essential role of changes
in alternative splicing in cancer progression means that virally
induced tumours may also be potentially treated by such drugs.
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