RNA virus infections and their effect on host alternative splicing

Abstract

It is evident that viral infection dramatically alters host gene expression, and these alterations have both pro- and anti-viral functions. While the effects of viral infection on transcription and translation have been comprehensively reviewed, less attention has been paid to the impact on alternative splicing of pre-messenger RNAs. Here we review salient examples of how viral infection leads to changes in alternative splicing and discuss how these changes impact infection.

Introduction

Most human protein-coding genes are transcribed into a precursor messenger RNAs (pre-mRNA), which have both introns and exons. The multistep process of excision of introns and ligation of exons to form a mature messenger RNA (mRNA) is known as pre-mRNA splicing, (aka splicing) (Berget et al., 1977; Chow et al., 1977). Exon-intron boundaries have conserved splice site sequences, primarily found at the 5’ and 3’ ends of introns, which are recognized by spliceosomes, macromolecular enzymes that catalyze intron removal and exon ligation (Fig. 1A). Splicing at many splice sites is not constitutive, resulting in the generation of transcript variants, this process is known as alternative pre-mRNA splicing or alternative splicing (Werner et al., 1992). Messenger RNA variants can be translated into different protein isoforms from a single gene, and these isoforms can have similar or dissimilar functions, and sometimes even antagonize each other. It is worthwhile noting that many non-coding RNA precursors are also alternatively spliced. According to the T2T-CHM13 human genome assembly, human cells have about 63,494 genes and out of these, 19,969 are protein-coding, but these give rise to 86,245 protein-coding transcripts (Nurk et al., 2022). This remarkable proteomic diversity is attributed to the alternative splicing observed in 95% of multi-exonic human protein-coding genes (Pan et al., 2008).

Figure 1. RNA splicing and types of alternative splicing.

(A) RNA splicing is catalyzed by the the spliceosome, a macromolecular enzyme that is formed on pre-mRNAs in a stepwise fashion. 1) U1 small nuclear ribonucleoprotein (snRNP) binds to the 5’ splice site, and SF1 and the U2AF heterodimer bind at the branch point sequence (BP), the polypyrimidine tract, and the 3’ splice site. 2) This early complex, which has been called E-complex or Commitment Complex, aided by auxiliary factors such as RNA helicases, recruits U2 snRNP to the branchpoint sequence. This forms what is known as the pre-spliceosome or Complex A. The U4•U5•U6 tri-snRNPs complex interacts with the pre-spliceosome to form the spliceosome, which undergoes multiple conformations eventually forming an active spliceosome. 3) Eventually, U1 and U4 snRNPs are released from the spliceosome. 4) The chemistry of splicing reactions involves two phosphoryl-transfer reactions that eventually produce the intron lariat and the ligated exons. (B) The different types of alternative splicing. (Grainger and Beggs, 2013; Marasco and Kornblihtt, 2022)

Alternative splicing can be classified into multiple types of events: intron retention (IR), exon skipping/inclusion of a cassette exon (CE), alternative 5’ splice site use (A5SS), alternative 3’ splice site use (A3SS), and mutually exclusive exons (MXE) (Fig. 1B) (Marasco and Kornblihtt, 2022).

Alternative splicing is critically important for every aspect of human biology including the function of our immune systems (Artemaki and Kontos, 2022; Baralle and Giudice, 2017; Liao and Garcia-Blanco, 2021; Marasco and Kornblihtt, 2022; Tang et al., 2013). Not surprisingly virus infections, which can cause massive disruption to cellular pathways, have been noted to alter alternative splicing in the host. This review is focused on providing an overview of how positive and negative sense RNA viruses differentially regulate alternative splicing Events (ASEs).

Viruses affect the splicing of the transcripts coding for RNA-binding proteins.

Viruses take over cellular pathways to sustain themselves in the host cells and given the importance of alternative splicing on varied cell functions it is not surprising that viral infections lead to dramatic changes in alternative splicing. Several viruses have been reported to target the post-transcriptional regulation of transcripts coding for splicing factors and spliceosome components, which in turn leads to differential alternative splicing of many host transcripts (Fig. 2). Enterovirus 71 (EV71), a member of the family Picornaviridae, affects the splicing of transcripts coding for U snRNP proteins (e.g., SNRNP70, SNRPA1, SNRPE), SR proteins (e.g., SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, and SRSF7), RNA helicases (e.g., DDX39B, DDX42, DDX5), and other pre-mRNA processing factors (e.g., PRPF18, PRPF3, PRPF40A) which affects their function and in turn, dysregulates alternative splicing (Li et al., 2020). Zika Virus (ZIKV, Flaviviridae family) infection has been found to enhance the exon skipping in SRSF2, HNRNPDL, and RBM39 transcripts, resulting in altered splicing of multiple target pre-mRNA. Interestingly, different isolates of ZIKV (Puerto Rican and Ugandan) were found to result in significantly different splicing of the previously mentioned transcripts, even though the levels of viral replication were comparable (Bonenfant et al., 2020). These RNA viruses alter global splicing patterns by modifying the processing of critical splicing factors.

Figure 2. Viruses target different aspects of the splicing pathway to alter splicing patterns in the host cell.

The left side of the illustration depicts the transcription, splicing, and translation of the proteins that play a role in RNA splicing in an uninfected cell. The right side presents examples of how virus infection affects alternative splicing. 1) Zika virus (ZIKV), and Enterovirus 71 (EV71) dysregulate the splicing of pre-mRNAs of genes coding for RNA binding proteins (RBPs) and splicing factors (SF). 2) Vesicular stomatitis (VSV), poliovirus (PV), and human immunodeficiency virus (HIV) block the nuclear translocation of SFs and RBPs. 3) Dengue virus (DENV) hijacks the active spliceosome via NS5 to deregulate splicing while Nsp16 of SARS CoV2 binds SRSF1 and SRSF2. The red lines represent dysregulated pathways leading to differential alternative splicing events.

Viral infections affect the subcellular localization of splicing factors.

During infection, viral factors interact with host proteins to sequester them into ectopic compartments, leading to cellular dysfunction (Avgousti et al., 2016). In the case of splicing factors, nuclear localization is the primary requisite for their function (Fig. 2). During poliovirus (family: Picornaviridae) infection, viral A2 protein (a protease) dysregulates the nuclear translocation of multiple RNA binding proteins (RBPs) (e.g., TIA1, TIA1R, PTB, and HuR), which affects the post-transcriptional processing of the pre-mRNAs of the host (Carpenter et al., 2014; Zhao et al., 2014). The blocked nuclear translocation of these RBPs results in increased exon 6 skipping in Fas mRNA (Álvarez et al., 2013), which encodes a Fas protein isoform that cannot induce apoptosis (Paronetto et al., 2014). During vesicular stomatitis virus (VSV; family: Rhabdoviridae) infection viral M1, M2, and M3 proteins dysregulate the nuclear localization of heterogeneous nuclear ribonucleoproteins (hnRNPs) such as redox factor 1 (Ref-1, aka ALYREF) and hnRNP H (Redondo et al., 2015). Also, the Human immunodeficiency virus (HIV), a member of Retroviridae family, changes the cellular localization of serine-arginine (SR) protein, SRSF2/SC-35 in addition to downregulating multiple SR proteins (SRp75, SRp55, SRp40, SRp30, and SRp20) levels and activity, which results in dysregulated RNA splicing (Fukuhara et al., 2006). Some RNA viruses alter alternative splicing decisions by modulating the subcellular localization of splicing factors.

Viral proteins and RNAs can interact with splicing factors and regulatory proteins to manipulate the splicing events of the host.

Splicing factors recognize splice sites as described in Figure 1. SARS-CoV-2 (a member of family Coronaviridae) nonstructural protein 16 (Nsp16) hijacks the splice site recognition mechanism by binding to U1 and U2 snRNAs (Fig. 2). This results in the global suppression of the splicing during the SARS-CoV-2 infection, (Srivastava et al., 2020; Wang et al., 2022). Overexpression of Nsp16 leads to intron retention of genes involved in innate immune responses such as RIG-I, IRF7, and ISG15(Banerjee et al., 2020). The numbers of differential ASEs in peripheral blood mononuclear cells (PBMCs) strongly correlated with disease severity; in patients with moderate disease 754 ASEs are altered, while in patients admitted to the Intensive care unit (ICU) 1246 ASEs differed significantly from those in PBMCs in the healthy control group (Wang et al., 2022).

Two different flaviviruses have been identified to take over the host splicing mechanism by interacting with the spliceosome components. Dengue virus (DENV) non-structural protein-5 (NS5) has two enzymatic activities: RNA-dependent RNA polymerase (RdRp) and methyl transferase (MTase). Being a RdRp, DENV NS5 is concentrated in viral RNA replication complexes in the cytoplasm (Uchil et al., 2006; van den Elsen et al., 2021); however, in some DENV infections up to 90% of total NS5 localizes to the nucleus of infected cells (Kumar et al., 2013). Although the importance of nuclear localization of NS5 in the virus life cycle is controversial (Pryor et al., 2007; Kumar et al., 2013; Fraser et al., 2014), the preponderance of the data suggests that NS5 may impact nuclear events directly. de Maio et al., showed that DENV NS5 is sequestered in nuclear speckles (interchromatin sites known for storing splicing factors). NS5 was found to be a part of the active splicing complex interacting with the pre-mRNA of different highly expressed genes such as HSPCB, Akt, RPS9, HPRT1, and TBP (Fig. 2). Hence, NS5 associates with U5snRNP to alter splicing patterns during DENV infection (de Maio et al., 2016). Consistent with this, the NS5 interactome includes components of the U5 short nuclear ribonucleoproteins (snRNP) complex such as CD2BP2, EFTUD2, and DDX23 (Wood et al., 2021). Apart from affecting the U5snRNP splicing complex DENV NS5 also targets RBM10 for proteasomal degradation (Fig. 2). RBM10 is known to regulate the splicing of apoptotic genes and degradation of RMB10 resuls in the upregulation of a pro-viral SAT1 isoform (Pozzi et al., 2020).

In addition to viral proteins viral non-coding RNAs can also modulate host alternative splicing patterns. Specifically, Zika virus (ZIKV) subgenomic flavivirus RNA (sfRNA) has been observed to interact and sponge off RBPs such as Phosphorylated Adaptor for RNA Export (PHAX), SF3B1/2, NMP1, and PPIH; dysregulating the alternative splicing. Overexpression of PHAX was found to negatively affect the ZIKV life cycle, suggesting that by sequestering PHAX, ZIKV creates a pro-viral state in host cell (Michalski et al., 2019).

We conclude that many RNA viruses interact with the splicing machinery to modulate the appearance of alternative splicing isoforms.

Viruses manipulate host RNA splicing to inhibit innate immunity in the host cell.

Multiple viruses incapacitate anti-viral responses by targeting gene transcription and protein degradation. Recently, alternative splicing of innate immune response genes has grabbed the attention of researchers (Fig. 3). RNA sequencing analysis of cells infected with various influenza virus (IAV)-strains (family: Orthomyxoviridae) exhibit changes in alternative splicing of key immune gene products. For instance, H3N2 IAV infection results in differential ASEs of mRNAs coding for TANK binding kinase 1 (TBK1), DNA Damage Inducible Transcript 3 (DDIT3), and Interferon Induced Protein 35 (IFI35), which regulate different aspects of innate immune responses (Fabozzi et al., 2018) (Fig. 3). H3N2 IAV is also known to inhibit the p53-mediated antiviral responses by altering the splicing of the TP53 gene transcript. Gene TP53 codes for the p53 protein which regulates multiple pathways such as cell cycle, DNA damage, apoptosis, immune responses, and inflammation. NS1 protein of H3N2 IAV interacts with Cleavage and Polyadenylation Specific Factor 4 (CPSF4) and promotes the intron 9 retention in TP53 transcript favoring accumulation of p53β and p53γ isoforms. This imbalance in p53 α, β, and γ isoforms in IAV-infected cell leads to the inhibition of p53-mediated interferon responses (Dubois et al., 2019). Infection with H1N1 IAV differentially regulates the splicing of other host factors, for example, splicing of the exoribonuclease, ERI2 short isoform is favored over the long isoform. The longer isoform of ERI2 has been shown to down-regulates the H1N1 replication if overexpressed, suggesting that ERI2 splicing is one-way HIN1 virus inhibits anti-viral mechanisms (Ashraf et al., 2020; Thompson et al., 2020). Respiratory Syncytial Virus (RSV; family: Pneumoviridae) is another respiratory virus that affects alternative splicing patterns to abrogate the anti-viral state. To downregulate type I interferon production, RSV favors the IKKγΔ isoform of Inhibitor of κB kinase gamma (IKKγ) (Liu et al., 2009; Xu et al., 2021)(Fig. 3). IKKγΔ protein lacks a coiled-coil domain important for protein-protein interactions that result in compromised NFκB signaling, which is essential for the IFN action (Hai et al., 2006).

Figure 3. Viruses take over the regulation of splicing to inhibit innate immune responses.

The illustration is showing how different viruses affect the specific checkpoints of innate immune pathways to create a pro viral state in host cell. RVFV tilts the balance of RIOK3 splicing towards RIOK3-X2, which affects RIOK3-RIG-I interaction. RSV increases the splicing of IKKγΔ isoform, which cannot interact with IKKα and β. This results in compromised inflammatory responses (e.g., TNFα). NCDV and SeV favor the splicing of tSTING-mini over tSTING-FL. Additionally, SeV and H3N2 IAV modify the splicing patterns of TBK1 to increase the levels of TBK1s. DENV has been reported to alters the splicing ratio of IKKε to IKKε sv1 and sv2. These isoforms of TBK1 and IKKε compromises the TBK1-IKKε-IKKγ complex formation. RSV, NCDV, SeV, H3N2 IAV, and DENV take advantage of alternative splicing mechanisms to down regulate type I interferon.

Two RNA viruses of Paramyxoviride family, New Castle disease virus (NCDV) and Sendai virus (SeV) alter the ratio of the two isoforms favoring the formation of the Tupia STING (tSTING)-mini coding variant (Fig. 3). The pro-viral effect of tSTING-mini was validated by its knockdown since NCDV and SeV showed reduced replication in tSTING-mini depleted cells (Xu et al., 2020). In addition to tSTING, SeV also alters the splicing patterns of TBK1 by upregulation of the splice variant TBK1s that lacks exons 3-6. TBK1 is a kinase that is central to interferon signaling (Zhou et al., 2020) and TBK1s disrupt the interaction between RIG-I and VISA by binding to RIG-I, which results in the inhibition of IRF3 signaling and type I IFN action (Deng et al., 2008; Gack et al., 2008; Xu et al., 2020).

Infection with the MP-12 attenuated strain of Rift Valley fever virus (RVFV), Phenuviridae family, results in multiple differential ASEs. Genes coding for transcripts with changes in splicing patterns were mostly categorized as being involved in immune responses, RNA processing, and transport. One important event is the differential A5SS in exon 8 of RIO kinase 3 (RIOK3)(Fig. 3). RVFV infection leads to increased abundance of the RIOK3-X2 isoform over the RIOK3-FL (full-length) isoform. RIOK3-FL protein plays a role in the IRF3 activation, while the truncated RIOK3-X2 leads to compromised IRF3 signaling resulting in reduced interferon signaling, but, paradoxically, uncontrolled inflammation (increased levels of TNFα and IL8) (Bisom et al., 2022; Havranek et al., 2019; White et al., 2021). DENV NS5 interacts with the spliceosome and alters the splicing of multiple innate immune factors such as IKKε and MX1 (de Maio et al., 2016).

Tip of the Iceberg

The inception of the concept of alternative splicing dates back to 1978 (Gilbert, 1978) and since then a large amount of work and effort has been put into understanding its intricate mechanisms. Nonetheless, a comprehensive study of alternative splicing events was not possible before RNA sequencing technology came into the picture in the year 2007. In the last decade, differential alternative splicing and its impact on disease conditions and the pathophysiology of infections have received intense attention. The understanding of how and why hundreds of splicing events are altered during the viral infection holds important keys to the mechanisms underlying disease biology. The studies briefly summarized here focus on how RNA viruses change alternative splicing patterns to create a pro-viral environment. A greater understanding of these events and their role will also lead to the identification of new targets and the design of better and more targeted therapies. These will not only target virus replication but should also regulate the host immune and inflammatory responses.