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

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 HHV-8, 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 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]. 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 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 SN2-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.

The basic splicing machinery

Figure 1
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.

Figure 1
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.

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 co-operative fashion to interfere with SR protein binding (Figure 2) [30].

Splicing factor interactions on a pre-mRNA

Figure 2
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.

Figure 2
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.

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 23000 genes, yet expresses over 90000 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, alternative ss selection, alternative promoter usage and alternative polyadenylation, with the former three being controlled by the splicing machinery (Figure 3).

Possible products of alternative splicing of a hypothetical gene

Figure 3
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.

Figure 3
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.

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 (mitogen-activated 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 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) [4144]. 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.

Table 1
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 
EBV LMP1, EBNA2 EBNA1, EBNA-LP, EBNA3A, EBNA3C, BARF1 BLLF1/BLLF, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, BZLF1, A73 STAT1, P450arom*, CD44SM, SRSF3, SRSF10*, hnRNPs* Burkitt's lymphoma, Hodgkin lymphoma, post-transplant lymphoma, X-linked lymphoproliferative syndrome, T-cell lymphomas, nasopharyngeal carcinoma 
KSHV/HHV8 LANA, vCyclin, vFLIP, Kaposins, K1, K15, vIRFs, vCCLs, vGPCR, vIL-6, vBCL-2, RTA, K-bZIP Kaposins, vCyclin.90, vIAP Kaposins (ORF K12), RTA?, K15, ORF73/ORF72/ORFK13, ORF50/ORFK8/ORFK8.1, ORF 59 ORF57, SRSF1, SRSF4, SRSF6, SRSF5, U2AF, hnRNP K Kaposi's sarcoma, primary effusion lymphomas, multicentric Castleman's disease 
HBV HBx  HBSP, PS SR*, SRPK* Hepatocellular carcinoma 
HCV    Hepatocellular carcinoma 
HPV E6, E7  E6, E7, E1, E4, L1 SRSF1, SRSF2, SRSF3 SRSF1, SRSF2, SRSF3, hnRNP A1 Cervical carcinoma, vaginal carcinoma, anal carcinoma, penile cancer, squamous cell carcinoma, actinic keratosis, head and neck squamous cell carcinomas 
HTLV Tax  X-region, env, rex, tax, mRNA: rof, tof Adult T-cell leukaemia 
Tumour virus Oncogenes Other genes involved in oncogenesis Viral alternative spliced genes/products Cellular controlled AS genes AS proteins involved Associated malignancies 
EBV LMP1, EBNA2 EBNA1, EBNA-LP, EBNA3A, EBNA3C, BARF1 BLLF1/BLLF, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, BZLF1, A73 STAT1, P450arom*, CD44SM, SRSF3, SRSF10*, hnRNPs* Burkitt's lymphoma, Hodgkin lymphoma, post-transplant lymphoma, X-linked lymphoproliferative syndrome, T-cell lymphomas, nasopharyngeal carcinoma 
KSHV/HHV8 LANA, vCyclin, vFLIP, Kaposins, K1, K15, vIRFs, vCCLs, vGPCR, vIL-6, vBCL-2, RTA, K-bZIP Kaposins, vCyclin.90, vIAP Kaposins (ORF K12), RTA?, K15, ORF73/ORF72/ORFK13, ORF50/ORFK8/ORFK8.1, ORF 59 ORF57, SRSF1, SRSF4, SRSF6, SRSF5, U2AF, hnRNP K Kaposi's sarcoma, primary effusion lymphomas, multicentric Castleman's disease 
HBV HBx  HBSP, PS SR*, SRPK* Hepatocellular carcinoma 
HCV    Hepatocellular carcinoma 
HPV E6, E7  E6, E7, E1, E4, L1 SRSF1, SRSF2, SRSF3 SRSF1, SRSF2, SRSF3, hnRNP A1 Cervical carcinoma, vaginal carcinoma, anal carcinoma, penile cancer, squamous cell carcinoma, actinic keratosis, head and neck squamous cell carcinomas 
HTLV Tax  X-region, env, rex, tax, mRNA: rof, tof Adult T-cell leukaemia 
*

Unknown mechanism.

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 RNA-binding phosphoprotein that regulates viral [4852] 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 [5161]. 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 EBV-positive 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 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 EBNA-1 proteins associate with SRSF1 [64,65] and other splicing-related 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 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 multi-membrane-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 [6668]. 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 [70,7376]. 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 pre-malignant 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 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.

Alternative splicing of the HPV16 E6 and E7 pre-mRNAs

Figure 4
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.

Figure 4
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.

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 concentration-dependent 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 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 non-cytopathic hepatotropic viruses. HBV is a partially double-stranded 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 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].

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 (Rex-response element) within viral RNAs via an N-terminal RNA-binding 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.

Splicing of the HTLV pX region and its regulation

Figure 5
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.

Figure 5
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.

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-molecular-mass 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 (histone deacetylase) inhibitors, CLK inhibitors, SRPK inhibitors, topoisomerase inhibitors, calmodulin kinase inhibitors, MAPK inhibitors and phosphatase inhibitors [121,122].

Table 2
Small-molecule names, types and molecular targets for inhibition of alternative splicing
Compound Drug type Systematic name Mechanism Reference 
IDC13 Indol derivative N-5,6-Dimethyl-5H-pyrido[3′,4′:4,5]pyrrolo[2,3-g]isoquinolin-10-yl-N′-ethyl-propane-1,3-diamine SR proteins* [138
IDC16 Indol derivative 10-Chloro-2,6-dimethyl-2H-pyrido[3′,4′,5]pyrrolo[2,3-g]isoquinoline Topo 1, SRSF1 [131
IDC78 Pyridocarbazole 1-Diethylamino-3(9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazol-1-ylamino)-propan-2-ol SR proteins* [138
NB-506 Indolocarbazole derivative 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 Topo 1 [134
KH-CB19 Dichloroindolyl enaminonitrile Ethyl-3-[(E)-2-amino-1-cyanoethenyl]-6,7-dichloro-1-methylindole-2-carboxylate CLK1, CLK4 [136
Leucettine L41 Leucettamine B derivative (5Z)-5-(1,3-Benzodioxol-5-yl)methylene-2-phenylamino-3,5-dihydro-4Himidazol-4-one CLKs, DYRKs [137
Isodiospyrin Diospyrin derivative 5-Hydroxy-6-(4-hydroxy-2-methyl-5,8-dioxonaphthalen-1-yl)-7-methylnaphthalene-1,4-dione Topo 1 [132,133
SRPIN340 Isonicotinamide derivative N-4-[2-Piperidino-5-(trifluoromethyl)phenyl]isonicotinamide SRPK1, SRPK2 [141
Chlorhexidine Biguanide 2,2′-(1,6-Hexanediyl)bis(1-{amino[(4-chlorophenyl)amino]methylene}guanidine) CLK2, CLK3, CLK4 [139
Camptothecin Alkaloid (4S)-4-Ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione Topo 1 [140
Compound Drug type Systematic name Mechanism Reference 
IDC13 Indol derivative N-5,6-Dimethyl-5H-pyrido[3′,4′:4,5]pyrrolo[2,3-g]isoquinolin-10-yl-N′-ethyl-propane-1,3-diamine SR proteins* [138
IDC16 Indol derivative 10-Chloro-2,6-dimethyl-2H-pyrido[3′,4′,5]pyrrolo[2,3-g]isoquinoline Topo 1, SRSF1 [131
IDC78 Pyridocarbazole 1-Diethylamino-3(9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazol-1-ylamino)-propan-2-ol SR proteins* [138
NB-506 Indolocarbazole derivative 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 Topo 1 [134
KH-CB19 Dichloroindolyl enaminonitrile Ethyl-3-[(E)-2-amino-1-cyanoethenyl]-6,7-dichloro-1-methylindole-2-carboxylate CLK1, CLK4 [136
Leucettine L41 Leucettamine B derivative (5Z)-5-(1,3-Benzodioxol-5-yl)methylene-2-phenylamino-3,5-dihydro-4Himidazol-4-one CLKs, DYRKs [137
Isodiospyrin Diospyrin derivative 5-Hydroxy-6-(4-hydroxy-2-methyl-5,8-dioxonaphthalen-1-yl)-7-methylnaphthalene-1,4-dione Topo 1 [132,133
SRPIN340 Isonicotinamide derivative N-4-[2-Piperidino-5-(trifluoromethyl)phenyl]isonicotinamide SRPK1, SRPK2 [141
Chlorhexidine Biguanide 2,2′-(1,6-Hexanediyl)bis(1-{amino[(4-chlorophenyl)amino]methylene}guanidine) CLK2, CLK3, CLK4 [139
Camptothecin Alkaloid (4S)-4-Ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione Topo 1 [140
*

Unknown mechanism.

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′-O-methylation, 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 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-2H-pyrido[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 tyrosine-phosphorylated and -regulated kinase) inhibitor, leucettine L41, has been described that inhibits phosphorylation of SRSF4, SRSF6 and SRSF7, and modulates alternative splicing in a cell-based 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.

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-dimethyl-5H-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 well-tolerated 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.

Abbreviations

     
  • 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

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.

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