Replication of positive-strand RNA viruses occurs in tight association with reorganized host cell membranes. In a concerted fashion, viral and cellular factors generate distinct organelle-like structures, designated viral replication factories. These virus-induced compartments promote highly efficient genome replication, allow spatiotemporal coordination of the different steps of the viral replication cycle, and protect viral RNA from the hostile cytoplasmic environment. The combined use of ultrastructural and functional studies has greatly increased our understanding of the architecture and biogenesis of viral replication factories. Here, we review common concepts and distinct differences in replication organelle morphology and biogenesis within the Flaviviridae family, exemplified by dengue virus and hepatitis C virus. We discuss recent progress made in our understanding of the complex interplay between viral determinants and subverted cellular membrane homeostasis in biogenesis and maintenance of replication factories of this virus family.

https://www.annualreviews.org/doi/pdf/10.1146/annurev-virology-100114-055007

Flaviviridae Replication Organelles: Oh, What a Tangled Web We Weave.

RNA Structure Duplications and Flavivirus Host Adaptation

Dengue (DENV) and Zika (ZIKV) viruses are clinically important members of the Flaviviridae family with an 11 kb positive strand RNA genome that folds to enable virus function. Here, we perform structure and interaction mapping on four DENV and ZIKV strains inside virions and in infected cells. Comparative analysis of SHAPE reactivities across serotypes nominates potentially functional regions that are highly structured, conserved, and contain low synonymous mutation rates. Interaction mapping by SPLASH identifies many pair-wise interactions, 40% of which form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared interactions between serotypes reveals a conserved macro-organization whereby interactions can be preserved at physical locations beyond sequence identities. We further observe that longer-range interactions are preferentially disrupted inside cells, and show the importance of new interactions in virus fitness. These findings deepen our understanding of Flavivirus genome organization and serve as a resource for designing therapeutics in targeting RNA viruses.

/pdf/structure-mapping-of-dengue-and-zika-viruses-reveals-3hqmk19xni.pdf

Structure mapping of dengue and Zika viruses reveals functional long-range interactions

In vivo structural characterization of the SARS-CoV-2 RNA genome identifies host proteins vulnerable to repurposed drugs.

Highly conserved noncoding RNA (ncRNA) elements in viral genomes and transcripts offer new opportunities to expand the repertoire of drug targets for the development of antiinfective therapy. Ligands binding to ncRNA architectures are able to affect interactions, structural stability or conformational changes and thereby block processes essential for viral replication. Proof of concept for targeting functional RNA by small molecule inhibitors has been demonstrated for multiple viruses with RNA genomes. Strategies to identify antiviral compounds as inhibitors of ncRNA are increasingly emphasizing consideration of drug-like properties of candidate molecules emerging from screening and ligand design. Recent efforts of antiviral lead discovery for RNA targets have provided drug-like small molecules that inhibit viral replication and include inhibitors of human immunodeficiency virus (HIV), hepatitis C virus (HCV), severe respiratory syndrome coronavirus (SARS CoV), and influenza A virus. While target selectivity remains a challenge for the discovery of useful RNA-binding compounds, a better understanding is emerging of properties that define RNA targets amenable for inhibition by small molecule ligands. Insight from successful approaches of targeting viral ncRNA in HIV, HCV, SARS CoV, and influenza A will provide a basis for the future exploration of RNA targets for therapeutic intervention in other viral pathogens which create urgent, unmet medical needs. Viruses for which targeting ncRNA components in the genome or transcripts may be promising include insect-borne flaviviruses (Dengue, Zika, and West Nile) and filoviruses (Ebola and Marburg). WIREs RNA 2016, 7:726-743. doi: 10.1002/wrna.1373 For further resources related to this article, please visit the WIREs website.

/pdf/small-molecules-targeting-viral-rna-5egkyijzr2.pdf

Small molecules targeting viral RNA.

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.

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

Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.

RNA structure duplication in the dengue virus 3′ UTR: redundancy or host specificity?

Zika virus (ZIKV) is an emerging flavivirus, mainly transmitted by mosquitoes, which represents a global health threat A common feature of flavivirus-infected cells is the accumulation of viral noncoding subgenomic RNAs by partial degradation of the viral genome, known as sfRNAs, involved in immune evasion and pathogenesis Although great effort is being made to understand the mechanism by which these sfRNAs function during infection, the picture of how they work is still incomplete In this study, we developed new genetic tools to dissect the functions of ZIKV RNA structures for viral replication and sfRNA production in mosquito and human hosts ZIKV infections mostly accumulate two kinds of sfRNAs, sfRNA1 and sfRNA2, by stalling genome degradation upstream of duplicated stem loops (SLI and SLII) of the viral 3' untranslated region (UTR) Although the two SLs share conserved sequences and structures, different functions have been found for ZIKV replication in human and mosquito cells While both SLs are enhancers for viral infection in human cells, they play opposite roles in the mosquito host The dissection of determinants for sfRNA formation indicated a strong cooperativity between SLI and SLII, supporting a high-order organization of this region of the 3' UTR Using recombinant ZIKV with different SLI and SLII arrangements, which produce different types of sfRNAs or lack the ability to generate these molecules, revealed that at least one sfRNA was necessary for efficient infection and transmission in Aedes aegypti mosquitoes Importantly, we demonstrate an absolute requirement of sfRNAs for ZIKV propagation in human cells In this regard, viruses lacking sfRNAs, constructed by deletion of the region containing SLI and SLII, were able to infect human cells but the infection was rapidly cleared by antiviral responses Our findings are unique for ZIKV, since in previous studies, other flaviviruses with deletions of analogous regions of the genome, including dengue and West Nile viruses, accumulated distinct species of sfRNAs and were infectious in human cells We conclude that flaviviruses share common strategies for sfRNA generation, but they have evolved mechanisms to produce different kinds of these RNAs to accomplish virus-specific functionsIMPORTANCE Flaviviruses are important emerging and reemerging human pathogens Understanding the molecular mechanisms for viral replication and evasion of host antiviral responses is relevant to development of control strategies Flavivirus infections produce viral noncoding RNAs, known as sfRNAs, involved in viral replication and pathogenesis In this study, we dissected molecular determinants for Zika virus sfRNA generation in the two natural hosts, human cells and mosquitoes We found that two RNA structures of the viral 3' UTR operate in a cooperative manner to produce two species of sfRNAs and that the deletion of these elements has a profoundly different impact on viral replication in the two hosts Generation of at least one sfRNA was necessary for efficient Zika virus infection of Aedes aegypti mosquitoes Moreover, recombinant viruses with different 3' UTR arrangements revealed an essential role of sfRNAs for productive infection in human cells In summary, we define molecular requirements for Zika virus sfRNA accumulation and provide new ideas of how flavivirus RNA structures have evolved to succeed in different hosts

/pdf/zika-virus-subgenomic-flavivirus-rna-generation-requires-ptfu8f8jf8.pdf

Zika Virus Subgenomic Flavivirus RNA Generation Requires Cooperativity between Duplicated RNA Structures That Are Essential for Productive Infection in Human Cells.

Dengue is the single most important human viral infection transmitted by insects. The function of the viral proteins andtheir interactions with the host cell is under exhaustive investigation with the aim of identifying antiviral strategies. Here,using recombinant full-length dengue virus genomes, carrying a fluorescent mCherry fused to capsid, we studied biophysicalproperties of the viral protein during one infectious cycle in living cells. Dengue virus capsid protein associates to differentcellular compartments but its function in these locations is largely unknown. We evaluated the diffusion of capsid inside the celland determined a higher effective diffusion coefficient in the cytoplasm than in the nucleus. Using advanced fluorescencecorrelation methods, including the recently developed two-dimensional pair correlation analysis, we constructed for the first timehigh resolution maps of capsid mobility in an infected cell. We observed that the motion of capsid in the nucleoplasm-nucleolusinterface was highly organized, indicating an obstacle in this interface. Although nucleoli are membraneless structures, theydisplayed liquid-liquid phase separation. Once inside nucleoli, the protein showed isotropic mobility, indicating free diffusion orimmobilized capsid inside these structures. This is the first study presenting spatial and temporal dynamics of the dengue viruscapsid protein during infection.

/pdf/dengue-virus-capsid-protein-dynamics-reveals-spatially-560ty43aas.pdf

Luana de Borba

Papers

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

RNA structure duplication in the dengue virus 3′ UTR: redundancy or host specificity?

Zika Virus Subgenomic Flavivirus RNA Generation Requires Cooperativity between Duplicated RNA Structures That Are Essential for Productive Infection in Human Cells.

Dengue Virus Capsid Protein Dynamics Reveals Spatially Heterogeneous Motion in Live-Infected-Cells.