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

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 functions.IMPORTANCE 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.

Information Encoded by the Flavivirus Genomes beyond the Nucleotide Sequence.

The genus Flavivirus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their successful propagation, flaviviruses use discrete structural genomic RNA elements to code for functional information by the establishment of dynamic networks of long-range RNA-RNA interactions that promote specific folding. These structural elements behave as true cis-acting, non-coding RNAs (ncRNAs) and have essential regulatory roles in the viral cycle. These include the control of the formation of subgenomic RNAs, known as sfRNAs, via the prevention of the complete degradation of the RNA genome. These sfRNAs are important in ensuring viral fitness. This work summarizes our current knowledge of the functions performed by the genome conformations and the role of RNA-RNA interactions in these functions. It also reviews the role of RNA structure in the production of sfRNAs across the genus Flavivirus, and their existence in related viruses.

Toward a Categorization of Virus-ncRNA Interactions in the World of RNA to Disentangle the Tiny Secrets of Dengue Virus.

In recent years, the function of noncoding RNAs (ncRNAs) as regulatory molecules of cell physiology has begun to be better understood. Advances in viral molecular biology have shown that host ncRNAs, cellular factors, and virus-derived ncRNAs and their interplay are strongly disturbed during viral infections. Nevertheless, the folding of RNA virus genomes has also been identified as a critical factor in regulating canonical and non-canonical functions. Due to the influence of host ncRNAs and the structure of RNA viral genomes, complex molecular and cellular processes in infections are modulated. We propose three main categories to organize the current information about RNA-RNA interactions in some well-known human viruses. The first category shows examples of host ncRNAs associated with the immune response triggered in viral infections. Even though miRNAs introduce a standpoint, they are briefly presented to keep researchers moving forward in uncovering other RNAs. The second category outlines interactions between virus-host ncRNAs, while the third describes how the structure of the RNA viral genome serves as a scaffold for processing virus-derived RNAs. Our grouping may provide a comprehensive framework to classify ncRNA-host-cell interactions for emerging viruses and diseases. In this sense, we introduced them to organize DENV-host-cell interactions.

Zika virus pathogenesis and current therapeutic advances.

Zika virus (ZIKV) is an emerging arthropod-borne flavivirus that, upon infection, results in teratogenic effects and neurological disorders. ZIKV infections pose serious global public health concerns, prompting scientists to increase research on antivirals and vaccines against the virus. These efforts are still ongoing as the pathogenesis and immune evasion mechanisms of ZIKV have not yet been fully elaborated. Currently, no specific vaccines or drugs have been approved for ZIKV; however, some are undergoing clinical trials. Notably, several strategies have been used to develop antivirals, including drugs that target viral and host proteins. Additionally, drug repurposing is preferred since it is less costly and takes less time than other strategies because the drugs used have already been approved for human use. Likewise, different platforms have been evaluated for the design of vaccines, including DNA, mRNA, peptide, protein, viral vectors, virus-like particles (VLPSs), inactivated-virus, and live-attenuated virus vaccines. These vaccines have been shown to induce specific humoral and cellular immune responses and reduce viremia and viral RNA both in vitro and in vivo. Importantly, most of these vaccines have entered clinical trials. Understanding the viral disease mechanism will provide better strategies for developing therapeutic agents against ZIKV. This review provides a comprehensive summary of the viral pathogenesis of ZIKV and current advancements in the development of vaccines and drugs against this virus.

RNA Structure Duplications and Flavivirus Host Adaptation.

Flaviviruses include a highly diverse group of arboviruses with a global distribution and a high human disease burden. Most flaviviruses cycle between insects and vertebrate hosts; thus, they are obligated to use different cellular machinery for their replication and mount different mechanisms to evade specific antiviral responses. In addition to coding for viral proteins, the viral genome contains signals in RNA structures that govern the amplification of viral components and participate in triggering or evading antiviral responses. In this review, we focused on new information about host-specific functions of RNA structures present in the 3' untranslated region (3' UTR) of flavivirus genomes. Models and conservation patterns of RNA elements of distinct flavivirus ecological groups are revised. An intriguing feature of the 3' UTR of insect-borne flavivirus genomes is the conservation of complex RNA structure duplications. Here, we discuss new hypotheses of how these RNA elements specialize for replication in vertebrate and invertebrate hosts, and present new ideas associating the significance of RNA structure duplication, small subgenomic flavivirus RNA formation, and host adaptation.