Bioinformatics analysis of selected aptamer sequences allows the identification of RNA tools for the functional analysis of West Nile virus genomic RNA elements

Abstract

The WNV is the etiological agent of recurrent outbreaks of febrile illness and encephalitis worldwide. Since the 1999 outbreak in the US, it is considered a global health threat by the WHO. The West Nile Virus (WNV) shares, with other RNA viruses, a compact RNA genome for storing all the required information for the completion of the infectious cycle. For this purpose, RNA viruses have developed an information storage system based on the use of discrete structural units that accomplish well-defined essential functions. This system complements and overlaps the protein coding one and provides an enormous plasticity to the viral genome. These structural/functional units can be conserved among closely related viruses and even among different isolates or strains. Therefore, they are considered potential therapeutic targets for fighting against viral infection. WNV is an enveloped, single-stranded positive RNA virus belonging to the genus Flavivirus (family Flaviviridae). It is a mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This genus comprises a large number of viruses, including important human pathogens as Dengue, Japanese encephalitis, Yellow fever or Zika virus, among others. The WNV genome is a single-stranded RNA molecule of ~11.000 nts, which contains a single open reading frame (ORF) flanked by essential untranslated regions (UTRs). The UTRs are rich in conserved structural RNA elements, which are functionally indispensable for the consecution of critical viral processes, as replication or translation. Such defined RNA structural elements participate in long-distant RNA-RNA interactions that bring close together both UTRs, thus enabling the acquisition of a closed-loop topology required for the achievement of the viral cycle. The WNV 3¿UTR can be subdivided into three autonomously folded regions, domains I-III, with the presence of duplications of structural cassettes (Figure 1). Domain I is located just downstream of the translation stop codon and appears as a hypervariable sequence followed by two conserved stem-loop domains (SL-I and -II) similar in sequence and structure (figure 1). Domain II is moderately conserved and contains a characteristic duplicated structure known as a dumbbell (DB); this is involved in the formation of a pseudoknot (PK) structural element. Domain III is defined by the highly conserved terminal genomic functional elements sHP (short hairpin) and 3¿SL. The functions of these structural elements have been studied in depth, and are essential for viral replication and the completion of the viral cycle. However, their role in WNV protein synthesis remains elusive. Therefore, determining their functional implications is a requisite to identify new potential targets for anti-WNV agents. In this context, the use of novel molecular tools based on nucleic acids, particularly RNA, for elucidating the role of different viral genomic regions has become a successful tactic recently included into the virologists¿ toolbox. Aptamers are single-stranded DNA or RNA molecules that bind in a very speci¿c and ef¿cient manner to their targets in a structure-dependent way. Aptamers are isolated by SELEX (Systematic Evolution of Ligands by EXponential enrichment), by which a highly heterogeneous initial population of DNA or RNA, usually composed of 1012 to 1015 variants, is subjected to iterative selection rounds against a speci¿c target.