Characterization of RVFV Nucleocapsid Protein Binding Sites on RNA by iCLIP-seq.

The nucleocapsid protein (N) in Rift Valley fever virus is an RNA-binding protein that functions in viral transcription, replication, and packaging. In this chapter, the method for studying protein-RNA interactions in context of viral infection using individual nucleotide resolution, cross-linking, immunoprecipitation, and sequencing (iCLIP-seq) is explained. The method is useful for identifying the interactions between both host and viral RNAs with N and can identify RNA motifs that interact with the protein of interest.

Using RNAseq to Uncover Transcriptional and Splicing Differences in Host Cells During Rift Valley Fever Virus Infection.

RNAseq is a valuable tool that can aid researchers in uncovering the transcriptional changes that occur when a viral pathogen infects a host cell. Viral infection will invariably cause differential expression of many genes, from transcription of mRNA to alternative splicing and degradation. This change in gene expression can be a result of immune activation or a direct activity of the virus to alter the host cell's environment to make it more favorable for viral replication. Studying the innate immune response to a pathogen can reveal which cellular pathways are active, indicating the steps that the host takes to halt viral infection, and detecting virus-mediated mRNA expression changes can help with identifying the pathways which may be exploited by the virus. Gene expression changes-both cell-caused and virus-caused-can be studied through RNAseq, helping to provide a clearer picture of the cellular changes that occur during viral infection. In this protocol, we outline methods to carry out mRNA sequencing in Rift Valley fever virus-infected cell cultures, from infection to library prep and analysis.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

RIOK3 and Its Alternatively Spliced Isoform Have Disparate Roles in the Innate Immune Response to Rift Valley Fever Virus (MP12) Infection.

Rift Valley fever virus (RVFV) is a pathogenic human and livestock RNA virus that poses a significant threat to public health and biosecurity. During RVFV infection, the atypical kinase RIOK3 plays important roles in the innate immune response. Although its exact functions in innate immunity are not completely understood, RIOK3 has been shown to be necessary for mounting an antiviral interferon (IFN) response to RVFV in epithelial cells. Furthermore, after immune stimulation, the splicing pattern for RIOK3 mRNA changes markedly, and RIOK3's dominant alternatively spliced isoform, RIOK3 X2, exhibits an opposite effect on the IFN response by dampening it. Here, we further investigate the roles of RIOK3 and its spliced isoform in other innate immune responses to RVFV, namely the NFκB-mediated inflammatory response. We find that while RIOK3 is important for negatively regulating this inflammatory pathway, its alternatively spliced isoform, RIOK3 X2, stimulates it. Overall, these data demonstrate that both RIOK3 and its X2 isoform have unique roles in separate innate immune pathways that respond to RVFV infection.

Time-Resolved Analysis of N-RNA Interactions during RVFV Infection Shows Qualitative and Quantitative Shifts in RNA Encapsidation and Packaging.

Rift Valley fever virus (RVFV) is a negative-sense, tripartite RNA virus that is endemic to Africa and the Arabian Peninsula. It can cause severe disease and mortality in humans and domestic livestock and is a concern for its potential to spread more globally. RVFV's nucleocapsid protein (N) is an RNA-binding protein that is necessary for viral transcription, replication, and the production of nascent viral particles. We have conducted crosslinking, immunoprecipitation, and sequencing (CLIP-seq) to characterize N interactions with host and viral RNAs during infection. In parallel, to precisely measure intracellular N levels, we employed multiple reaction monitoring mass spectrometry (MRM-MS). Our results show that N binds mostly to host RNAs at early stages of infection, yielding nascent virus particles of reduced infectivity. The expression of N plateaus 10 h post-infection, whereas the intracellular viral RNA concentration continues to increase. Moreover, the virions produced later in infection have higher infectivity. Taken together, the detailed examination of these N-RNA interactions provides insight into how the regulated expression of N and viral RNA produces both infectious and incomplete, noninfectious particles.

The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing.

In recent years, transcriptome profiling studies have identified changes in host splicing patterns caused by viral invasion, yet the functional consequences of the vast majority of these splicing events remain uncharacterized. We recently showed that the host splicing landscape changes during Rift Valley fever virus MP-12 strain (RVFV MP-12) infection of mammalian cells. Of particular interest, we observed that the host mRNA for Rio Kinase 3 (RIOK3) was alternatively spliced during infection. This kinase has been shown to be involved in pattern recognition receptor (PRR) signaling mediated by RIG-I like receptors to produce type-I interferon. Here, we characterize RIOK3 as an important component of the interferon signaling pathway during RVFV infection and demonstrate that RIOK3 mRNA expression is skewed shortly after infection to produce alternatively spliced variants that encode premature termination codons. This splicing event plays a critical role in regulation of the antiviral response. Interestingly, infection with other RNA viruses and transfection with nucleic acid-based RIG-I agonists also stimulated RIOK3 alternative splicing. Finally, we show that specifically stimulating alternative splicing of the RIOK3 transcript using a morpholino oligonucleotide reduced interferon expression. Collectively, these results indicate that RIOK3 is an important component of the mammalian interferon signaling cascade and its splicing is a potent regulatory mechanism capable of fine-tuning the host interferon response.

Tra2beta-Dependent Regulation of RIO Kinase 3 Splicing During Rift Valley Fever Virus Infection Underscores the Links Between Alternative Splicing and Innate Antiviral Immunity.

Rift Valley fever virus (RVFV) is an emerging pathogen that has potential to cause severe disease in humans and domestic livestock. Propagation of RVFV strain MP-12 is negatively impacted by the actions of RIOK3, a protein involved in the cellular immune response to viral infection. During RVFV infection, RIOK3 mRNA is alternatively spliced to produce an isoform that correlates with the inhibition of interferon ß signaling. Here, we identify splicing factor TRA2-ß (also known as TRA2beta and hTRA2-ß) as a key regulator governing the relative abundance of RIOK3 splicing isoforms. Using RT-PCR and minigenes, we determined that TRA2-ß interaction with RIOK3 pre-mRNA was necessary for constitutive splicing of RIOK3 mRNA, and conversely, lack of TRA2-ß engagement led to increased alternative splicing. Expression of TRA2-ß was found to be necessary for RIOK3's antiviral effect against RVFV. Intriguingly, TRA2-ß mRNA is also alternatively spliced during RVFV infection, leading to a decrease in cellular TRA2-ß protein levels. These results suggest that splicing modulation serves as an immune evasion strategy by RVFV and/or is a cellular mechanism to prevent excessive immune response. Furthermore, the results suggest that TRA2-ß can act as a key regulator of additional steps of the innate immune response to viral infection.

C/D box snoRNAs in viral infections: RNA viruses use old dogs for new tricks.

C/D box snoRNAs (SNORDs) are a highly expressed class of non-coding RNAs. Besides their well-established role in rRNA modification, C/D box snoRNAs form protein complexes devoid of fibrillarin and regulate pre-mRNA splicing and polyadenylation of numerous genes. There is an emerging body of evidence for functional interactions between RNA viruses and C/D box snoRNAs. The infectivity of some RNA viruses depends on enzymatically active fibrillarin, and many RNA viral proteins associate with nucleolin or nucleophosmin, suggesting that viruses benefit from their cytosolic accumulation. These interactions are likely reflected by morphological changes in the nucleolus, often leading to relocalization of nucleolar proteins and ncRNAs to the cytosol that are a characteristic feature of viral infections. Knock-down studies have also shown that RNA viruses need specific C/D box snoRNAs for optimal replication, suggesting that RNA viruses benefit from gene expression programs regulated by SNORDs, or that viruses have evolved "new" uses for these humble ncRNAs to advance their prospects during infection.

Transcriptome profiling in Rift Valley fever virus infected cells reveals modified transcriptional and alternative splicing programs.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development.

RNA Encapsidation and Packaging in the Phleboviruses.

The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5-7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.

Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.

Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications.

Inhibition of Rift Valley fever virus replication and perturbation of nucleocapsid-RNA interactions by suramin.

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. There are currently no proven safe and effective treatment options for RVFV infection. Inhibition of RNA binding to RVFV nucleocapsid protein (N) represents an attractive antiviral therapeutic strategy because several essential steps in the RVFV replication cycle involve N binding to viral RNA. In this study, we demonstrate the therapeutic potential of the drug suramin by showing that it functions well as an inhibitor of RVFV replication at multiple stages in human cell culture. Suramin has been used previously to treat trypanosomiasis in Africa. We characterize the dynamic and cooperative nature of N-RNA binding interactions and the dissociation of high-molecular-mass ribonucleoprotein complexes using suramin, which we previously identified as an N-RNA binding inhibitor in a high-throughput screen. Finally, we elucidate the molecular mechanism used by suramin in vitro to disrupt both specific and nonspecific binding events important for ribonucleoprotein formation.

Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

Characterization of RNA aptamers directed against the nucleocapsid protein of Rift Valley fever virus.

Nucleocapsid protein (N) is an essential RNA binding protein in many RNA viruses. During replication, N protein encapsidates viral genomic and antigenomic RNA, but not viral mRNA or other cellular RNAs. To discriminate between different species of RNA in a host cell, it is likely that N interacts with specific sequences and/or secondary structures on its target RNA. In this study, we explore the RNA binding properties of N using both natural and artificially selected RNAs as ligands. We found that N binds to RNAs that resemble the terminal panhandle structures of RVFV genomic and antigenomic RNA. Furthermore, we used SELEX to isolate RNA aptamers that bound N with high affinity and determined that N specifically recognizes and binds to GAUU and pyrimidine/guanine motifs. Interestingly, BLAST analysis revealed the presence of these motifs within the coding region of the viral genome, suggesting that N may interact with non-terminal viral RNA sequences during replication. Finally, the aptamer RNAs were used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies.

The roles of transcription and genotoxins underlying p53 mutagenesis in vivo.

Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication.

The 5' UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5' untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The "winner" viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication.

A 5'UTR-spliced mRNA isoform is specialized for enhanced HIV-2 gag translation.

Full-length unspliced genomic RNA plays critical roles in HIV replication, serving both as mRNA for the synthesis of the key viral polyproteins Gag and Gag-Pol and as genomic RNA for encapsidation into assembling viral particles. We show that a second gag mRNA species that differs from the genomic RNA molecule by the absence of an intron in the 5' untranslated region (5'UTR) is produced during HIV-2 replication in cell culture and in infected patients. We developed a cotransfection system in which epitopically tagged Gag proteins can be traced back to their mRNA origins in the translation pool. We show that a disproportionate amount of Gag is translated from 5'UTR intron-spliced mRNAs, demonstrating a role for the 5'UTR intron in the regulation of gag translation. To further characterize the effects of the HIV-2 5'UTR on translation, we fused wild-type, spliced, or mutant leader RNA constructs to a luciferase reporter gene and assayed their translation in reticulocyte lysates. These assays confirmed that leaders lacking the 5'UTR intron increased translational efficiency compared to that of the unspliced leader. In addition, we found that removal or mutagenesis of the C-box, a pyrimidine-rich sequence located in the 5'UTR intron and previously shown to affect RNA dimerization, also strongly influenced translational efficiency. These results suggest that the splicing of both the 5'UTR intron and the C-box element have key roles in regulation of HIV-2 gag translation in vitro and in vivo.

Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA.

The packaging signal (psi) of human immunodeficiency virus type 2 (HIV-2) is present in the 5' noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5'-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3' side of pal (GCUCC-3') is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5' side of pal (5'-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5' side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5' side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.

Regulation of primate lentiviral RNA dimerization by structural entrapment.

BACKGROUND: Genomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1) element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1. To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, here we compare the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Prior studies from several groups have employed a variety of constructs and experimental conditions. RESULTS: Although some idiosyncratic differences in the dimerization details were observed, we find unifying principles in the regulation strategies of the three viral RNAs through long- and short-range base pairing interactions. Presentation and efficacy of dimerization through SL1 depends strongly upon the formation or dissolution of the lower stem of SL1 called stem B. SL1 usage may also be down-regulated by long-range interactions involving sequences between SL1 and the first codons of the gag gene. CONCLUSION: Despite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1.

HIV-2 RNA dimerization is regulated by intramolecular interactions in vitro.

Genomic RNA dimerization is an essential process in the retroviral replication cycle. In vitro, HIV-2 RNA dimerization is mediated at least in part by direct intermolecular interaction at stem-loop 1 (SL1) within the 5'-untranslated leader region (UTR). RNA dimerization is thought to be regulated via alternate presentation and sequestration of dimerization signals by intramolecular base-pairings. One of the proposed regulatory elements is a palindrome sequence (pal) located upstream of SL1. To investigate the role of pal in the regulation of HIV-2 dimerization, we randomized this motif and selected in vitro for dimerization-competent and dimerization-impaired RNAs. Energy minimization folding analysis of these isolated sequences suggests the involvement of pal region in several short-distance intramolecular interactions with other upstream and downstream regions of the UTR. Moreover, the consensus predicted folding patterns indicate the altered presentation of SL1 depending on the interactions of pal with other regions of RNA. The data suggest that pal can act as a positive or negative regulator of SL1-mediated dimerization and that the modulation of base-pairing arrangements that affect RNA dimerization could coordinate multiple signals located within the 5'-UTR.