Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

Abstract

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed.

A highly sensitive strand-specific multiplex RT-qPCR assay for quantitation of Zika virus replication

“…Increased specificity, i.e. beyond 1000:1 (+):(-) RNA, is likely unnecessary as the (+):(-) RNA ratio during infection is within our assay's range of specificity, where a ratio of 10:1-100:1 excess positivestrand RNA has been reported (3)(4)(5)(6). Furthermore, decreased sensitivity would impede negative-strand detection at early time points and render the assay less able to quantify early events in viral replication.…”

“…Given that there is reported to be up to 100-fold excess of positivestrand compared with negative-strand viral RNA during ZIKV infection, we first determined whether the primer concentrations needed for accurate detection of the negative-strand in multiplex PCR would need optimization (3)(4)(5)(6). In a multiplex qPCR assay, it is important to verify that the amplification of high-abundance targets does not interfere with detection of lowabundance targets by depleting reagents (e.g.…”

“…Like all positive-sense RNA viruses, ZIKV replication proceeds through a negative-strand replication intermediate. New positive-strands are synthesized from the negativestrand template, and positive-strand synthesis generally outnumbers negative-strand synthesis by ~10-100 fold (3)(4)(5)(6). Negative-strand RNA detection is therefore the gold standard for detection of ZIKV replication.…”

PERK Is Critical for Alphavirus Nonstructural Protein Translation

“…For example, flaviviruses contain a positive-sense genomic RNA with a 5′ cap. Upon viral entry, viral translation occurs through a canonical cap-dependent process [ 58 ], similar to alphaviruses. In contrast, RVFV being a negative sense RNA virus requires the transcription of its genetic material prior to translation occurring.…”

The 5′ and 3′ Untranslated Regions of the Japanese Encephalitis Virus (JEV): Molecular Genetics and Higher Order Structures

“…The capsid-coding region hairpin (cHP) element lies within the coding region and aids in start codon recognition and viral replication (Clyde et al, 2008). The 5 UAR along with 5 downstream AUG region (DAR) (Friebe et al, 2011) and the 5 cyclization sequence (5 cCS) (Villordo and Gamarnik, 2009;Gebhard et al, 2011) cause genome circularization by hybridizing with their counterparts in the 3 UTR (i.e., 3 UAR, 3 DAR, and 3 cCS) (Hahn et al, 1987;Hodge et al, 2019). Besides the viral genome cyclization-related sequences, 3 UTR has a panel of stem-loop structures that halt XRN1 exoribonuclease digestion.…”