Short Direct Repeats in the 3′ Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production

Abstract

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation. IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5ʹ–3ʹ exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3ʹ-UTR. The 3ʹ-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Structures and Functions of the 3′ Untranslated Regions of Positive-Sense Single-Stranded RNA Viruses Infecting Humans and Animals

“…Moreover, the deletion of the four miR-142-3p binding sites in the 3 ′ UTR of EEEV result in FIGURE 7 | The biogenesis of sfRNA in WNV-infected cells. The primary sequences (RCS3, CS3, RCS2, CS2) and higher order structures (SL-II, SL-IV, DB1, DB2, PK1, PK2, PK3) of the WNV 3 ′ UTR are required for production of sfRNAs (Pijlman et al, 2008;Funk et al, 2010;Zhang et al, 2020).…”

“…Recent research shows that RCS3, CS3, RCS2, and CS2 in WNV 3′ UTR are also involved in the production of their corresponding upstream sfRNAs. Among these sequences, RCS3 increases the binding affinity of xrRNA and Xrn1 and stabilizes the three-dimensional structure of the xrRNA (Zhang et al, 2020 ). Furthermore, the PK is critical for xrRNA function.…”

An African Tick Flavivirus Forming an Independent Clade Exhibits Unique Exoribonuclease-Resistant RNA Structures in the Genomic 3′-Untranslated Region

“…Characteristic secondary structures of both the 5′-UTR and the 3′-UTR are required for genome cyclization, and interaction of the UTRs is essential for viral replication and translation [16][17][18][19][20][21][22] . During avivirus infection, shorter subgenomic avivirus RNAs (sfRNAs) are produced in addition to viral genomic RNAs [23][24][25][26][27][28] . The production of sfRNA is common to all aviviruses, and is associated with viral pathogenicity, host adaptation, and immune evasion [26][27][28][29] .…”

“…During avivirus infection, shorter subgenomic avivirus RNAs (sfRNAs) are produced in addition to viral genomic RNAs [23][24][25][26][27][28] . The production of sfRNA is common to all aviviruses, and is associated with viral pathogenicity, host adaptation, and immune evasion [26][27][28][29] . These sfRNAs originate from the 3′-UTR of the avivirus genome and result from partial degradation of viral genomic RNA by the cellular 5′-3′ exoribonuclease Xrn1, which degrades 5′-monophosphorylated RNAs in the cytoplasm 23 .…”

“…The xrRNA duplication observed in vector-borne aviviruses is related to host adaptation without reduction of viral tness during host switching between invertebrates and vertebrates25,29 . The rst xrRNA of West Nile virus, when mutated to disrupt its structure, signi cantly led to reduction in its pathogenicity,indicating that this xrRNA plays a role in virulence to vertebrate hosts28 . Interestingly, deletion of the second xrRNA in Dengue virus led to an increase in viral replication in mosquito cells, whereas deletion of both xrRNAs reduced viral replication in mammalian cells29 .…”