West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5'CS/3'CSI (conserved sequence) interaction, a 5'UAR/3'UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5'UARs capable of forming the 5'/3' viral RNA interaction were replicative. In contrast, WNV containing a 5'UAR mutation that abolished the 5'/3' viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5'/3' RNA interaction. The 5'UAR/3'UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5'UAR/3'UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3'UAR could alternate between pairing with the 5'UAR or with the 3' end of the flaviviral genome. Therefore, the 5'UAR/3'UAR pairing may release the 3' end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.     

Core protein-mediated 5'-3' annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5' and 3' terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA-RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5'-3' genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5'-3' complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5'-3' RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein - besides its function in viral particle formation - might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.     

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.     

Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3' stem-loop structure

Dengue virus (DEN) is a major public health problem worldwide and causes a spectrum of diseases, for which no antiviral treatments exist. Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) complementary to the DEN 5' stem-loop (5'SL) and to the DEN 3' cyclization sequence (3'CS) inhibit DEN replication, presumably by blocking critical RNA-RNA or RNA-protein interactions involved in viral translation and/or RNA synthesis. Here, a third P-PMO, complementary to the top of the 3' stem-loop (3'SLT), inhibited DEN replication in BHK cells. Using a novel DEN2 reporter replicon and a DEN2 reporter mRNA, we determined that the 5'SL P-PMO inhibited viral translation, the 3'CS P-PMO blocked viral RNA synthesis but not viral translation, and the 3'SLT P-PMO inhibited both viral translation and RNA synthesis. These results show that the 3'CS and the 3'SL domains regulate DEN translation and RNA synthesis and further demonstrate that P-PMOs are potentially useful as antiviral agents.     

Genetic analysis of West Nile virus containing a complete 3'CSI RNA deletion

We report a genetic interplay among three pairs of long-distance RNA interactions that are involved in West Nile virus (WNV) genome cyclization and replication: 5′CS/3′CSI (conserved sequence), 5′UAR/3′UAR (upstream AUG region), and 5′DAR/3′DAR (downstream AUG region). Deletion of the complete 3′CSI element is lethal for WNV replication, but the replication of the 3′CSI deletion virus could be rescued by second site mutations. Functional analysis, using a genome-length RNA and replicon, mapped the compensatory mutations to the 5′UAR/3′UAR and 5′DAR/3′DAR regions. Biochemical analysis showed that the 3′CSI deletion abolished the 5′ and 3′ RNA interaction of the genome; the compensatory mutations could partially restore the 5′ and 3′ genome cyclization. These results demonstrate, for the first time, that a flavivirus without 3′CSI could restore genome cyclization and viral replication through enhancement of the 5′UAR/3′UAR and 5′DAR/3′DAR interactions.     

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.     

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5′-terminal 190 nucleotides and the 3′-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5′CS/3′CSI and 5′UAR/3′UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3′-terminal nucleotides of the genome (position − 8 to − 16) to be single-stranded. Viral NS5 binds specifically to the 5′-terminal stem-loop (SL1) of the genomic RNA. The 5′SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5′SL1 RNA interaction facilitate NS5 binding to the 3′ end of the genome for the initiation of viral minus-strand RNA synthesis.     

Composition of the sequence downstream of the dengue virus 5' cyclization sequence (dCS) affects viral RNA replication

RNA replication of dengue virus (DENV) requires an RNA-RNA mediated circularization of the viral genome, which includes at least three sets of complementary RNA sequences on both ends of the genome. The 5′ and the 3′ untranslated regions form several additional RNA elements that are involved in regulation of translation and required for RNA replication. Communication between the genomic termini results in a structural reorganization of the RNA elements, forming a functional RNA panhandle structure. Here we report that the sequence composition downstream of the 5′ CS element in the capsid gene, designated as downstream CS (dCS) sequence - but not the capsid protein - also influences the ability of the viral genome to circularize and hence replicate by modulating the topology of the 5′ end. These results provide insights for the design of reporter sub-genomic and genomic mosquito-borne flavivirus constructs and contribute to the understanding of viral RNA replication.     

The flavivirus-conserved penta-nucleotide in the 3' stem-loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation

A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3' untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3' UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5'-CACAG-3' located at the top of the 3' stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication.     

Co-selection of West Nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance RNA/RNA base pairings

West Nile virus (WNV) genome cyclization is mediated by two pairs of long-distance RNA/RNA interactions: the 5′CS/3′CSI (conserved sequence) and the 5′UAR/3′UAR (upstream AUG region) base pairings. Antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), designed to interfere with the 5′CS/3′CSI or 5′UAR/3′UAR base pairings, were previously shown to inhibit WNV. In this study, we selected and characterized WNVs resistant to a PPMO targeting the 3′UAR (3′UAR-PPMO). All resistant viruses accumulated one-nucleotide mutations within the 3′UAR, leading to a single-nucleotide mismatch or a weakened base-pairing interaction with the 3′UAR-PPMO. Remarkably, a one-nucleotide mutation within the 5′UAR was correspondingly co-selected; the 5′UAR mutation restored the base pairing with the 3′UAR mutation. Mutagenesis of WNV demonstrated that the single-nucleotide change within the 3′UAR-PPMO-target site conferred the resistance. RNA binding analysis indicated that the single-nucleotide change reduced the ability of 3′UAR-PPMO to block the RNA/RNA interaction required for genome cyclization. The results suggest a mechanism by which WNV develops resistance to 3′UAR-PPMO, through co-selection of the 5′UAR and 3′UAR, to create a mismatch or a weakened base-pairing interaction with the PPMO, while maintaining the 5′UAR/3′UAR base pairings.     

The majority of the nucleotides in the top loop of the genomic 3' terminal stem loop structure are cis-acting in a West Nile virus infectious clone

The flavivirus genome RNA terminates with a conserved 3' stem loop (SL) structure that was shown to be essential for virus replication. A stretch of conserved nts is located in the top loop (TL) of this structure. Mutation of the TL nts (5' ACAGUGC 3') in a WNV infectious clone indicated that 3 of the 7 TL nts (5' ACAGUGC 3') are critical for virus replication. Mutation of 3 of the other nts reduced the efficiency of virus replication. The four 5' TL nts are conserved in both mosquito- and tick-borne flavivirus genomes, while the TL 3' C is conserved in mosquito-borne viruses. The conservation of two or three G-C base pairs in the TL flanking sequences suggests that a stable stem is necessary for precise presentation of the TL sequence. The TL may participate in RNA as well as protein interactions.     

Genome cyclization as strategy for flavivirus RNA replication

Long-range and local RNA-RNA contacts in viral RNA genomes result in tertiary structures that modulate the function of enhancers, promoters, and silencers during translation, RNA replication, and encapsidation. In the case of flaviviruses, the presence of inverted complementary sequences at the 5' and 3' ends of the genome mediate long-range RNA interactions and RNA cyclization. The circular conformation of flavivirus genomes was demonstrated to be essential for RNA amplification. New ideas about the mechanisms by which circular genomes participate in flavivirus replication have emerged in the last few years. Here, we will describe the latest information about cis-acting elements involved in flavivirus genome cyclization, RNA promoter elements required for viral polymerase recognition, and how these elements together coordinate viral RNA synthesis.     

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.     

Adaptation of Venezuelan equine encephalitis virus lacking 51-nt conserved sequence element to replication in mammalian and mosquito cells

Replication of alphaviruses strongly depends on the promoters located in the plus- and minus-strands of virus-specific RNAs. The most sophisticated promoter is encoded by the 5' end of the viral genome. This RNA sequence is involved in the initiation of translation of viral nsPs, and synthesis of both minus- and plus-strands of the viral genome. Part of the promoter, the 51-nt conserved sequence element (CSE), is located in the nsP1-coding sequence, and this limits the spectrum of possible mutations that can be performed. We designed a recombinant Venezuelan equine encephalitis virus genome, in which the promoter and nsP1-coding sequences are separated. This modification has allowed us to perform a wide variety of genetic manipulations, without affecting the amino acid sequence of the nsPs, and to further investigate 51-nt CSE functioning. The results of this study suggest a direct interaction of the amino terminal domain of nsP2 with the 5' end of the viral genome.     

Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.