Subgenomic flaviviral RNAs: What do we know after the first decade of research

Slonchak, Andrii; Khromykh, Alexander A.

doi:10.1016/j.antiviral.2018.09.006

Cited by 97 publications

(128 citation statements)

References 133 publications

(272 reference statements)

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“…A partial exonucleolytic digest by Xrn1 is common among flaviviruses, resulting in the generation of subgenomic flavivirus RNA (sfRNA) (reviewed in reference 37). Xrn1 also degrades HCV RNA, which is counteracted by the binding of miR-122 (38)(39)(40). We have further shown previously that miR-122 is capable of functional binding to the internal UTR copy (34).…”

Section: Internal Initiation By Hcv Polymerasementioning

confidence: 55%

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Schult

Nattermann

Lauber

et al. 2019

J Virol

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Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5= untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or templateswitching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNAdependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

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Section: Internal Initiation By Hcv Polymerasementioning

confidence: 55%

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Schult

Nattermann

Lauber

et al. 2019

J Virol

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“…In this study we have identified and structurally characterized xrRNAs in all genera of Flaviviridae, one of the largest families of RNA viruses, expanding on the known distribution of these RNA structures within Flaviviridae (23). Based on our computational, structural, and biochemical data, we propose that the characteristics of these new xrRNAs require a division of the previously proposed class 1 xrRNA (38) into two distinct subclasses: subclass 1a (comprising MBFV, in particular MVEV, ZIKV, WNV, YFV, etc.…”

Section: Discussionmentioning

confidence: 99%

“…1A) (22). During infection, a subset of flaviviral genomes is targeted to the cellular RNA decay machinery, then processively degraded in a 5′ to 3′ direction by Xrn1, until the enzyme halts at an xrRNA in the 3′ untranslated region (UTR) (19,23). The fold of the xrRNA is sufficient for this function, and no accessory proteins nor chemical modification of the RNA are required.…”

Section: Introductionmentioning

confidence: 99%

“…The fold of the xrRNA is sufficient for this function, and no accessory proteins nor chemical modification of the RNA are required. The xrRNA and the protected downstream RNA comprise a non-coding subgenomic flaviviral RNA (sfRNA) that accumulates in the cell and performs several important functions for the virus (2,19,(23)(24)(25)(26), including inhibiting host antiviral response (27,28), affecting viral transmissibility (29)(30)(31)(32)(33), and disrupting the host mRNA program (34,35).…”

Section: Introductionmentioning

confidence: 99%

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A new subclass of exoribonuclease resistant RNA found in multipleFlaviviridaegenera

Szucs

Nichols

Jones

et al. 2020

Preprint

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Viruses have developed innovative strategies to exploit the cellular machinery and overcome the host antiviral defenses, often using specifically structured RNA elements. Examples are found in flaviviruses; during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5’ to 3’ exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease resistant RNA structure (xrRNA) located in the viral genome’s 3’untranslated region (UTR). Although known to exist in several Flaviviridae genera the full distribution and diversity of xRNAs in this virus family was unknown. Using the recent high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNA in the Pegivirus, Pestivirus, and Hepacivirus genera. We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Our findings thus require adjustments of previous xrRNA classification schemes and expand on the previously known distribution of the xrRNA in Flaviviridae, indicating their widespread distribution and illustrating their importance.IMPORTANCEThe Flaviviridae comprise one of the largest families of positive sense single stranded (+ssRNA) and it is divided into the Flavivirus, Pestivirus, Pegivirus, and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika Virus, Dengue Virus, and Powassan Virus. In these, a part of the virus’s RNA twists up into a very special three-dimensional shape called an xrRNA that blocks the ability of the cell to “chew up” the viral RNA. Hence, part of the virus’ RNA remains intact, and this protected part is important for viral infection. This was known to occur in Flaviviruses but whether it existed in the other members of the family was not known. In this study, we not only identified a new subclass of xrRNA found in Flavivirus but also in the remaining three genera. The fact that this process of viral RNA maturation exists throughout the entire Flaviviridae family makes it clear that this is an important but underappreciated part of the infection strategy of these diverse human pathogens.

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“…sfRNAs have been associated with pathogenic outcomes and cytopathicity during infection (Funk et al 2010;Chang et al 2013;Walker et al 2013;Donald et al 2016;Filomatori et al 2017;Junglen et al 2017). Although their full set of functions remains an active area of investigation (Slonchak and Khromykh 2018) sfRNAs have been shown to bind a number of host proteins (Bidet et al 2014;Schnettler et al 2014;Manokaran et al 2015;Moon et al 2015;Goertz et al 2019;Michalski et al 2019) suggesting they affect several processes during infection including inhibiting the interferon response (Schuessler et al 2012) and suppressing RNAi pathways in insects (Schnettler et al 2012;Schnettler et al 2014;Moon et al 2015). Furthermore, there is evidence that sfRNAs are involved in switching between insect vectors and mammalian hosts (Filomatori et al 2017;de Borba et al 2019), enhancing transmission by mosquitoes (Pompon et al 2017;Yeh and Pompon 2018;Goertz et al 2019;Slonchak et al 2020), and altering the cell's mRNA decay program (Moon et al 2012;Michalski et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

Jones

Steckelberg

Szucs

et al. 2020

Preprint

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During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3′ untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5′ end of the xrRNA.Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3′ UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3′ UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg 2+ ions, and a C + -G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

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Subgenomic flaviviral RNAs: What do we know after the first decade of research

Cited by 97 publications

References 133 publications

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

A new subclass of exoribonuclease resistant RNA found in multipleFlaviviridaegenera

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

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