Claudia V. Filomatori

Functional RNA Elements in the Dengue Virus Genome

Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5′ and 3′ UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5′ end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3′ end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle.

Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

ABSTRACT The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template.

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3′ end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3′-UTR, we found that the RNA-RNA interaction mediated by 5′-3′-hybridization was able to release the silencing effect of the 3′-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity.

Dengue virus RNA structure specialization facilitates host adaptation

Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3’UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

ABSTRACT The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5′ untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.

Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

ABSTRACT The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3′ untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells

The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3’UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3’UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3’UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3’UTRs with possible implications in virulence and viral transmission.

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production

ABSTRACT Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. IMPORTANCE Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process.