Zika virus dumbbell-1 structure is critical for sfRNA presence and cytopathic effect during infection.

All flaviviruses contain conserved RNA structures in the 3' untranslated region (3' UTR) that are important for flavivirus RNA replication, translation, and pathogenesis. Flaviviruses like Zika virus (ZIKV) contain multiple conserved RNA structures in the viral 3' UTR, including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus positive-strand genome replication, but the functional role of the flavivirus DB-1 structure and the mechanism by which it contributes to viral pathogenesis are not known. Using the recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones, termed ZIKV-TL.PK and ZIKV-p.2.5', which disrupt DB-1 tertiary folding. We found that viral positive-strand genome replication of both ZIKV DB-1 mutant clones is similar to wild-type (WT) ZIKV, but ZIKV DB-1 mutants exhibit significantly decreased cytopathic effect due to reduced caspase-3 activation. We next show that ZIKV DB-1 mutants exhibit decreased levels of sfRNA species compared to ZIKV-WT during infection. However, ZIKV DB-1 mutant 3' UTRs exhibit unchanged sfRNA biogenesis following XRN1 degradation in vitro. We also found that ZIKV DB-1 mutant virus (ZIKV-p.2.5') exhibited enhanced sensitivity to type I interferon treatment, and both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality due to tissue-specific attenuated viral replication in brain tissue of interferon type I/II receptor knockout mice. We propose that the flavivirus DB-1 RNA structure maintains sfRNA levels during infection despite maintained sfRNA biogenesis, and these results indicate that ZIKV DB-dependent maintenance of sfRNA levels support caspase-3-dependent, cytopathic effect, type I interferon resistance, and viral pathogenesis in mammalian cells and in a ZIKV murine model of disease. IMPORTANCE The group of viruses termed flaviviruses cause important disease throughout the world and include dengue virus, Zika virus, Japanese encephalitis virus, and many more. All of these flaviviruses have highly conserved RNA structures in the untranslated regions of the virus genome. One of the shared RNA structures, termed the dumbbell region, is not well studied, but mutations in this region are important for vaccine development. In this study, we made structure-informed targeted mutations in the Zika virus dumbbell region and studied the effect on the virus. We found that Zika virus dumbbell mutants are significantly weakened or attenuated due to a decreased ability to produce non-coding RNA that is needed to support infection, support virus-induced cell death, and support escape from the host immune system. These data show that targeted mutations in the flavivirus dumbbell RNA structure may be an important approach to develop future vaccine candidates.

The 21st Annual Meeting of the Rocky Mountain Virology Association.

Nestled within the Rocky Mountain National Forest, 114 scientists and students gathered at Colorado State University's Mountain Campus for this year's 21st annual Rocky Mountain National Virology Association meeting. This 3-day retreat consisted of 31 talks and 30 poster presentations discussing advances in research pertaining to viral and prion diseases. The keynote address provided a timely discussion on zoonotic coronaviruses, lessons learned, and the path forward towards predicting, preparing, and preventing future viral disease outbreaks. Other invited speakers discussed advances in SARS-CoV-2 surveillance, molecular interactions involved in flavivirus genome assembly, evaluation of ethnomedicines for their efficacy against infectious diseases, multi-omic analyses to define risk factors associated with long COVID, the role that interferon lambda plays in control of viral pathogenesis, cell-fusion-dependent pathogenesis of varicella zoster virus, and advances in the development of a vaccine platform against prion diseases. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations.

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication.

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

Accurate design of translational output by a neural network model of ribosome distribution.

Synonymous codon choice can have dramatic effects on ribosome speed and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs. Here, we have modeled this variation in translation elongation by using a feed-forward neural network to predict the ribosome density at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and characterized large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of translation speeds predicted with our model. Levels of the fluorescent protein in budding yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo; that this information can be harnessed to design coding sequences; and that control of translation elongation alone is sufficient to produce large quantitative differences in protein output.

Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Reversible lineage-specific priming of human embryonic stem cells can be exploited to optimize the yield of differentiated cells.

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here, we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible, as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore, the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM, thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential, which can be modulated by culture conditions in a predictive and quantitative manner.

Human embryonic stem cell-derived hematopoietic cells maintain core epigenetic machinery of the polycomb group/Trithorax Group complexes distinctly from functional adult hematopoietic stem cells.

Hematopoietic cells derived from human embryonic stem cells (hESCs) have a number of potential utilities, including the modeling of hematological disorders in vitro, whereas the use for cell replacement therapies has proved to be a loftier goal. This is due to the failure of differentiated hematopoietic cells, derived from human pluripotent stem cells (hPSCs), to functionally recapitulate the in vivo properties of bona fide adult hematopoietic stem/progenitor cells (HSPCs). To better understand the limitations of differentiation programming at the molecular level, we have utilized differential gene expression analysis of highly purified cells that are enriched for hematopoietic repopulating activity across embryonic, fetal, and adult human samples, including in vivo explants of human HSPCs 8-weeks post-transplantation. We reveal that hESC-derived hematopoietic progenitor cells (eHPCs) fail to express critical transcription factors which are known to govern self-renewal and myeloid/lymphoid development and instead retain the expression of Polycomb Group (PcG) and Trithorax Group (TrxG) factors which are more prevalent in embryonic cell types that include EZH1 and ASH1L, respectively. These molecular profiles indicate that the differential expression of the core epigenetic machinery comprising PcGs/TrxGs in eHPCs may serve as previously unexplored molecular targets that direct hematopoietic differentiation of PSCs toward functional HSPCs in humans.

Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Kaiso regulates Znf131-mediated transcriptional activation.

Kaiso is a dual-specificity POZ-ZF transcription factor that regulates gene expression by binding to sequence-specific Kaiso binding sites (KBS) or methyl-CpG dinucleotide pairs. Kaiso was first identified as a binding partner for the epithelial cell adhesion regulator p120(ctn). The p120(ctn)/Kaiso interaction is reminiscent of the beta-catenin/TCF interaction and several studies have suggested that Kaiso is a negative regulator of the Wnt/beta-catenin TCF signaling pathway. To gain further insight into Kaiso's function, we performed a yeast two-hybrid screen using the Kaiso POZ domain as bait. This screen identified the POZ-ZF protein, Znf131, as a Kaiso-specific binding partner. GST pull-down assays confirmed that the interaction is mediated via the POZ domain of each protein, and co-immunoprecipitation experiments further supported an in vivo Kaiso-Znf131 interaction. Using a Cyclic Amplification and Selection of Targets (CAST) approach, we identified the 12-base pair DNA palindrome sequence GTCGCR-(X)(n)-YGCGAC as a potential Znf131 binding element (ZBE). In vitro studies using electrophoretic mobility shift assay (EMSA) demonstrated that Znf131 binds the ZBE via its zinc finger domain. Znf131 DNA-binding specificity was confirmed using competition assays and ZBE mutational analyses. An artificial promoter-reporter construct containing four tandem copies of the ZBE was constructed and used to assess Znf131 transcriptional properties. We observed dose-dependent transcriptional activation of this artificial promoter-reporter by Znf131 in both epithelial and fibroblast cells, suggesting that Znf131 is a transcriptional activator. Kaiso overexpression significantly decreased the Znf131-mediated transcriptional activation, and interestingly, co-expression of the Kaiso-specific interaction partner p120(ctn) relieved Kaiso's inhibition of Znf131-mediated transcriptional activation. These findings indicate that Znf131 is a transcriptional activator, a less common function of POZ-ZF proteins, that is negatively regulated by its heterodimerization partner Kaiso.

Nuclear trafficking of the POZ-ZF protein Znf131.

Znf131 is a member of the BTB/POZ family of transcription factors with roles in development and carcinogenesis. Like many members of this protein family, Znf131 displays robust nuclear localization in cultured cells, but the mechanism(s) of Znf131 nuclear trafficking is unknown. Here, we report the mechanism of Znf131 nuclear localization. Visual inspection of the Znf131 amino acid sequence revealed three basic regions (BR-1, -2 and -3) with the potential to serve as nuclear localization signals (NLS). Of the three basic regions, only BR-1 functioned independently to efficiently target heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. However, a Znf131 truncation mutant containing BR-2 and BR-3 efficiently targeted heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. Mutational analysis of full-length GFP-tagged Znf131 revealed that loss of any one BR alone did not prevent Znf131 nuclear localization. This apparent redundancy in NLS activity was due to the fact that intact BR-1 or BR-2 alone could target full-length Znf131 to nuclei. Consequently, simultaneous mutation of BR-1 and BR-2 abolished full-length Znf131 nuclear localization. Therefore, BR-1 and BR-2 are functional NLSs for Znf131 and as such are designated NLS-1 and NLS-2. Finally, wild type Znf131, and not a Znf131 NLS-defective mutant (NLS-1m/NLS-2m) interacted preferentially with the nuclear import receptor Importin-alpha3 in vitro.

The catenin p120ctn inhibits Kaiso-mediated transcriptional repression of the beta-catenin/TCF target gene matrilysin.

The POZ-zinc finger transcription factor Kaiso was first identified as a specific binding partner for the Armadillo catenin and cell adhesion cofactor, p120ctn. Kaiso is a unique POZ protein with bi-modal DNA-binding properties; it associates with a sequence-specific DNA consensus Kaiso binding site (KBS) or methylated CpG dinucleotides, and regulates transcription of artificial promoters containing either site. Interestingly, the promoter of the Wnt/beta-catenin/TCF target gene matrilysin possesses two conserved copies of the KBS, which suggested that Kaiso might regulate matrilysin expression. In this study, we demonstrate using chromatin immunoprecipitation analysis that Kaiso associates with the matrilysin promoter in vivo. Minimal promoter assays further confirmed that Kaiso specifically repressed transcription of the matrilysin promoter; mutation of the KBS element or RNAi-mediated depletion of Kaiso abrogated this effect. More importantly, Kaiso blocked beta-catenin-mediated activation of the matrilysin promoter. Consistent with our previous findings, both Kaiso-DNA binding and Kaiso-mediated transcriptional repression of the matrilysin promoter were inhibited by overexpression of wild-type p120ctn, but not by a p120ctn mutant exhibiting impaired nuclear import. Collectively, our data establish Kaiso as a sequence-specific transcriptional repressor of the matrilysin promoter, and suggest that p120ctn and beta-catenin act in a synergistic manner, via distinct mechanisms, to activate matrilysin expression.

Nuclear import of the BTB/POZ transcriptional regulator Kaiso.

Kaiso is a BTB/POZ transcription factor that functions in vitro as a transcriptional repressor of the matrix metalloproteinase gene matrilysin and the non-canonical Wnt signaling gene Wnt-11, and as an activator of the acetylcholine-receptor-clustering gene rapsyn. Similar to other BTB/POZ proteins (e.g. Bcl-6, PLZF, HIC-1), endogenous Kaiso localizes predominantly to the nuclei of mammalian cells. To date, however, the mechanism of nuclear import for most POZ transcription factors, including Kaiso, remain unknown. Here, we report the identification and characterization of a highly basic nuclear localization signal (NLS) in Kaiso. The functionality of this NLS was verified by its ability to target a heterologous beta-galactosidase/green-fluorescent-protein fusion protein to nuclei. The mutation of one positively charged lysine to alanine in the NLS of full-length Kaiso significantly inhibited its nuclear localization in various cell types. In addition, wild-type Kaiso, but not NLS-defective Kaiso, interacted directly with the nuclear import receptor Importin-alpha2 both in vitro and in vivo. Finally, minimal promoter assays using a sequence-specific Kaiso-binding-site fusion with luciferase as reporter demonstrated that the identified NLS was crucial for Kaiso-mediated transcriptional repression. The identification of a Kaiso NLS thus clarifies the mechanism by which Kaiso translocates to the nucleus to regulate transcription of genes with diverse roles in cell growth and development.