Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Distinct chikungunya virus polymerase palm subdomains contribute to virus replication and virion assembly.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus (CHIKV) has established the importance of the nsP4 residue cysteine 483 in maintaining viral genetic fidelity. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the Coxsackievirus B3 3 polymerase. We rescued each variant in both mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to polymerase host-specific replication and genetic stability. Surprisingly, in mosquito cells, these variants in the ring finger and palm domain were replication competent and produced viral structural proteins, but they were unable to produce infectious progeny, indicating a yet uncharacterized role for the polymerase in viral assembly. Finally, we have identified additional residues in the nsP4 palm domain that influence the genetic diversity of the viral progeny, potentially via an alteration in NTP binding and/or discrimination by the polymerase. Taken together, these studies highlight that distinct nsP4 subdomains regulate multiple processes of the alphavirus life cycle, placing nsP4 in a central role during the switch from RNA synthesis to packaging and assembly.

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.

RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

TMEM41B is a pan-flavivirus host factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration.

Small non-coding RNAs have emerged as key modulators of viral infection. However, with the exception of hepatitis C virus, which requires the liver-specific microRNA (miRNA)-122, the interactions of RNA viruses with host miRNAs remain poorly characterized. Here, we used crosslinking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries, critically depended on the interaction of cellular miR-17 and let-7 with the viral 3' UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de-repression of cellular miR-17 targets, thereby altering the host transcriptome. These findings generalize the concept of RNA virus dependence on cellular miRNAs and connect virus-induced miRNA sequestration to host transcriptome regulation.

Fidelity Variants and RNA Quasispecies.

By now, it is well established that the error rate of the RNA-dependent RNA polymerase (RdRp) that replicates RNA virus genomes is a primary driver of the mutation frequencies observed in RNA virus populations-the basis for the RNA quasispecies. Over the last 10 years, a considerable amount of work has uncovered the molecular determinants of replication fidelity in this enzyme. The isolation of high- and low-fidelity variants for several RNA viruses, in an expanding number of viral families, provides evidence that nature has optimized the fidelity to facilitate genetic diversity and adaptation, while maintaining genetic integrity and infectivity. This chapter will provide an overview of what fidelity variants tell us about RNA virus biology and how they may be used in antiviral approaches.

Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles.

UNLABELLED: Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE: Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion.

UNLABELLED: To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions. IMPORTANCE: Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.

Emergence and transmission of arbovirus evolutionary intermediates with epidemic potential.

The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapitulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary trajectories can be predicted and studied in the short term before new variants displace currently circulating strains.

Alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models.

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.

The magnitude of dengue virus NS1 protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model.

There are conflicting data on the relationship between the level of secreted NS1 (sNS1), viremia, and disease severity upon dengue virus (DENV) infection in the clinical setting, and therefore, we examined this relationship in the widely accepted AG129 mouse model. Because of the failure of a routinely used NS1 detection kit to detect sNS1 of the mouse-adapted DENV2 strain, we screened 15 previously undescribed NS1 monoclonal antibodies and developed a robust capture enzyme-linked immunosorbent assay (ELISA) with detection sensitivity at the low nanogram level (0.2 ng/ml) using recombinant baculovirus-expressed sNS1 as well as sNS1 that was immunoaffinity purified from the various DENV2 strains employed in this study. Using this test, we demonstrated that increased viremia paralleled severe pathologies; however, sNS1 level did not correlate with viremia or severity. Furthermore, among the DENV2 strains that were tested, the level of NS1 secretion did not correspond to virus replication rate in vitro, at the cellular level. Together, our data indicate that the magnitude of NS1 secretion appears to be strain dependent and does not correlate with viral virulence in the AG129 mouse model.

Expression and immunoaffinity purification of recombinant dengue virus 2 NS1 protein as a cleavable SUMOstar fusion.

Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S∗-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S∗-tag increasing protein solubility and secretion compared with a non-S∗-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S∗-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S∗-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions.

Determinants of homodimerization specificity in histidine kinases.

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.