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.

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.

Different tertiary interactions create the same important 3D features in a distinct flavivirus xrRNA.

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure.

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease.

The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure that is responsible for ZIKV sfRNA production forms a complex fold that is likely found in many pathogenic flaviviruses. Mutations that disrupt the structure affect exonuclease resistance in vitro and sfRNA formation during infection. The complete ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies features that may modulate function in diverse flaviviruses.

Structured RNAs that evade or confound exonucleases: function follows form.

Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi's sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation.

The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids.

Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA-DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity.

A conserved motif in Tetrahymena thermophila telomerase reverse transcriptase is proximal to the RNA template and is essential for boundary definition.

The ends of linear chromosomes are extended by telomerase, a ribonucleoprotein complex minimally consisting of a protein subunit called telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a short template region of TER into telomeric DNA. Proper assembly of TERT and TER is essential for telomerase activity; however, a detailed understanding of how TERT interacts with TER is lacking. Previous studies have identified an RNA binding domain (RBD) within TERT, which includes three evolutionarily conserved sequence motifs: CP2, CP, and T. Here, we used site-directed hydroxyl radical probing to directly identify sites of interaction between the TERT RBD and TER, revealing that the CP2 motif is in close proximity to a conserved region of TER known as the template boundary element (TBE). Gel shift assays on CP2 mutants confirmed that the CP2 motif is an RNA binding determinant. Our results explain previous work that established that mutations to the CP2 motif of TERT and to the TBE of TER both permit misincorporation of nucleotides into the growing DNA strand beyond the canonical template. Taken together, these results suggest a model in which the CP2 motif binds the TBE to strictly define which TER nucleotides can be reverse transcribed.

The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA.

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65(CTD) is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65(CTD) interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem-loop IV.

Single-molecule analysis of telomerase structure and function.

The telomerase ribonucleoprotein is a specialized reverse transcriptase required to maintain protective chromosome end-capping structures called telomeres. In most cells, telomerase is not active and the natural shortening of telomeres with each round of DNA replication ultimately triggers cell growth arrest. In contrast, the presence of telomerase confers a high level of renewal capacity upon rapidly dividing cells. Telomerase is aberrantly activated in 90% of human cancers and thus represents an important target for anticancer therapeutics. However, the naturally low abundance of telomerase has hampered efforts to obtain high-resolution models for telomerase structure and function. To circumvent these challenges, single-molecule techniques have recently been employed to investigate telomerase assembly, structure, and catalysis.

The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis.

Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3' end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation.

Assembly of complex RNAs by splinted ligation.

Mechanistic studies of RNA enzymes (ribozymes) and ribonucleoprotein (RNP) complexes such as the ribosome and telomerase, often seek to characterize RNA structural features, either dynamic or static, and relate these properties to specific catalytic functions. Many experimental techniques that probe RNA structure-function relationships rely upon site-specific incorporation of chemically modified ribonucleotides into the RNA of interest, often in the form of chemical cross-linkers to probe for sites of protein-RNA interaction or small organic fluorophores to measure dynamic structural properties of RNAs. The ability to arbitrarily modify any RNA molecule has been greatly enabled by modern RNA synthesis techniques; however, there remains a practical size limitation (~70 bases). Consequently, experimental approaches involving specific chemical modifications of larger RNAs require the use of RNA ligation methods. The aim of this chapter is to describe a general approach for covalently joining multiple site-specifically modified RNA fragments, drawing from our fluorescence-based structural studies of telomerase RNA as an example.