The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce non-coding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exoribonuclease using structured RNAs called xrRNAs. Here, we demonstrate 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.

Flaviviruses are emerging human pathogens and worldwide health threats. During infection, a pathogenic, subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5’→3’ host cell exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly-conserved nucleotides. The RNA’s three-dimensional topology creates a ring-like conformation with the 5’ end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.

Internal ribosome entry site (IRES) RNAs initiate protein synthesis in eukaryotic cells by a noncanonical cap-independent mechanism. IRESes are critical for many pathogenic viruses, but efforts to understand their function are complicated by the diversity of IRES sequences as well as by limited high-resolution structural information. The intergenic region (IGR) IRESes of the Dicistroviridae viruses are powerful model systems to begin to understand IRES function. Here we present the crystal structure of a Dicistroviridae IGR IRES domain that interacts with the ribosome's decoding groove. We find that this RNA domain precisely mimics the transfer RNA anticodonmessenger RNA codon interaction, and its modeled orientation on the ribosome helps explain translocation without peptide bond formation. When combined with a previous structure, this work completes the first high-resolution description of an IRES RNA and provides insight into how RNAs can manipulate complex biological machines.Canonical eukaryotic translation initiation is a complex, multistep process in which a modified nucleotide cap on the 5′ end of the mRNA is necessary for protein factor-dependent recruitment of the 40S (small) ribosomal subunit 1 . The subunit scans the message and recognizes the AUG initiation codon, which leads to GTP hydrolysis, initiation factor protein release, and 60S (large) subunit binding to form the 80S ribosome-mRNA complex. The assembled 80S ribosome, containing an initiator tRNA in the P site, begins translation through eukaryotic elongation factor (eEF) 1A-mediated delivery of an aminoacylated tRNA into the A site. Subsequent peptide bond formation in the ribosome's peptidyl transferase site is followed by eEF 2-catalyzed translocation, and the ribosome enters the elongation phase of protein synthesis (Fig. 1a). Thus, canonical cap-dependent translation initiation is driven by the action of many protein factors and begins in the P site from an AUG codon in good context; then translocation occurs after the initial peptide bond is made.In contrast to the canonical cap-dependent pathway, an important alternate mechanism is internal initiation of translation. This mechanism depends on specific cis-acting RNA sequences called IRESes 2,3 . There is great diversity in IRES sequence, secondary structure and requirements for protein factors, but all IRESes recruit, position and activate the proteinmaking machinery without recognizing the cap or the 5′ end of the mRNA. IRESes are critical for the infection of many pathogenic viruses and may be important regulatory elements in gene expression 2 . Insight into the mechanism of many IRESes has been provided by a variety of approaches, but details of their RNA structure-based function remain incomplete. (refs. 4-6 ), and both contact the ribosome over the E site and through the large subunit's L1 stalk 4,5,7,8 . Furthermore, cryo-EM reconstructions of these IRES RNAs bound to the ribosome indicate that both may undergo subtle structural rearrangement during the course of preini...

Canonical cap-dependent translation initiation requires a large number of protein factors that act in a stepwise assembly process. In contrast, internal ribosomal entry sites (IRESs) are cis-acting RNAs that in some cases completely supplant these factors by recruiting and activating the ribosome using a single structured RNA. Here we present the crystal structures of the ribosome-binding domain from a Dicistroviridae intergenic region IRES at 3.1 angstrom resolution, providing a view of the prefolded architecture of an all-RNA translation initiation apparatus. Docking of the structure into cryo-electron microscopy reconstructions of an IRES-ribosome complex suggests a model for ribosome manipulation by a dynamic IRES RNA.In eukaryotes, there are two known mechanisms for the initiation of protein synthesis (Fig. 1A). The canonical mechanism requires a modified nucleotide cap on the 5′ end of the mRNA, which is recognized by an initiation factor protein (eIF4E). This protein recruits other factors that assemble the ribosome on the mRNA in a stepwise process (1). In contrast, internal initiation of translation does not require a cap or recognition of the mRNA 5′ end. Rather, structured RNA sequences called internal ribosomal entry sites (IRESs) recruit and activate the translation machinery, functionally replacing many protein factors (2). IRESs are essential for infection by many medically and economically important viruses such as hepatitis C (HCV), hepatitis A, polio, foot-and-mouth disease, rhinovirus, coxsackievirus-B3, and HIV-1 (3). IRESs also drive the translation of eukaryotic mRNAs, encoding factors involved in development, growth regulation, apoptosis, transcription, translation, and other important cellular processes (3). The molecular rules underlying this RNA structure-driven mechanism remain elusive.Ideal model systems for understanding IRES RNA-driven translation are the mechanistically streamlined intergenic region (IGR) IRESs of the virus family Dicistroviridae (4). The IGR IRESs drive the association of the ribosomal subunits without any of the protein factors that comprise the canonical translation initiation apparatus (Fig. 1A) (5). Hence, this one structured RNA (molecular size ∼66 kD) supplants over 1000 kD of structured initiation factor proteins, operating as an all-RNA translation initiation apparatus (6-9). The full-length IGR IRES folds in solution into two structurally independent domains (10-13). The larger domain (regions 1 and 2, Fig. 1A and fig. S1) is the ribosome-binding domain. It folds into a compact structure (10) that binds directly to the 40S subunit (10,12,13). Cryo-electron microscopy (cryo-EM) reconstructions of an IGR IRES bound to the ribosome reveal that the IGR IRES binds over the mRNA-binding groove, making contact to and changing the structure of both ribosomal subunits (40S and 60S) (14). However, these cryo-EM structures do not reveal the structure of the IRES, how the IRES structure creates a ribosome-binding site, or which IRES structural features speci...