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

Barnard, Trisha R.; Wang, Alex B.; Sagan, Selena M.

doi:10.1016/j.jviromet.2022.114556

Cited by 3 publications

(5 citation statements)

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“…We next investigated the relationship between viral RNA replication and dsRNA. Using a strand-specific RT-qPCR assay, we quantified positive- and negative-strand viral RNA accumulation during infection () [31]. We found that ZIKV PR induced significantly more positive- and negative-strand viral RNA than ZIKV PR -NS3+5 Mut between 9 and 18 h post-infection, suggesting that ZIKV PR initiates viral RNA replication more efficiently than ZIKV PR -NS3+5 Mut ().…”

Section: Resultsmentioning

confidence: 99%

“…ZIKV RNA was quantified based on a standard curve of in vitro -transcribed positive- and negative-sense genomes and was normalized to GAPDH. The RT-qPCR assay for negative strand quantitation was specific to 1000-fold excess positive-strand RNA [31].…”

Section: Methodsmentioning

confidence: 99%

“…Five hundred nanograms of total RNA was subject to strand-specific RT-qPCR as previously described [31]. ZIKV RNA was quantified based on a standard curve of in vitro -transcribed positive- and negative-sense genomes and was normalized to GAPDH.…”

Section: Methodsmentioning

confidence: 99%

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Zika virus NS3 and NS5 proteins determine strain-dependent differences in dsRNA accumulation in a host cell type-dependent manner

Barnard

Wang

Sagan

2023

Journal of General Virology

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For positive-sense RNA viruses, initiation of viral RNA replication represents a major target of antiviral responses to infection. Despite this, the interplay between viral replication and the innate antiviral response at early steps in the Zika virus (ZIKV) life cycle is not well understood. We have previously identified ZIKV isolates with differing levels of dsRNA accumulation, ZIKVPR (high dsRNA per infected cell) and ZIKVCDN (low dsRNA per infected cell), and we hypothesized that we could use reverse genetics to investigate how host and viral factors contribute to the establishment of viral RNA replication. We found that both the ZIKV NS3 and NS5 proteins as well as host factors were necessary to determine the dsRNA accumulation phenotype. Additionally, we show that dsRNA correlates with viral negative-strand RNA measured by strand-specific RT-qPCR, suggesting that dsRNA is an accurate readout of viral RNA replication. Interestingly, although we did not observe NS3- and NS5-dependent differences in cells with defects in interferon (IFN) production, differences in RNA accumulation precede induction of the IFN response, suggesting that RNA sensing pathways or intrinsic restriction factors may differentially restrict ZIKV in an NS3- and NS5-dependent manner. This work expands our understanding of the interplay of early steps of viral RNA replication and the induction of the innate antiviral response to ZIKV infection.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Zika virus NS3 and NS5 proteins determine strain-dependent differences in dsRNA accumulation in a host cell type-dependent manner

Barnard

Wang

Sagan

2023

Journal of General Virology

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“…Flavivirus RNA replication is a two-step process that involves: 1) negative-strand RNA synthesis, which uses the single-stranded positive-sense genomic RNA as a template and results in a double-stranded (ds) RNA replicative intermediate; and subsequently, 2) positive-strand RNA synthesis, using the dsRNA replicative intermediate as a template. Notably, viral RNA replication is a tightly regulated process, with positive-strand genomic RNAs outnumbering negative-strands on the order of approximately 50:1 during infection [ 56 ]. Genome cyclization is implicated in loading of the NS5 RdRp on the 3´ terminus of the positive-strand genomic RNA for negative-strand RNA synthesis.…”

Section: How Do Rna Structures Direct Viral Rna Replication?mentioning

confidence: 99%

“…The major difference between negative- and positive-strand RNA synthesis relates to the nature of the template RNA, which is single-stranded positive-sense genomic RNA and the dsRNA replicative intermediate, respectively. Although each end of the dsRNA replicative intermediate is biochemically similar and possesses all the features needed for the core replicase to act, there is a clear preference for the initiation of positive-strand RNA synthesis relative to negative-strand RNA synthesis [ 56 , 57 , 76 ]. As discussed previously, since the specificity needed to maintain this preference does not come from the viral proteins involved, it is likely that features of the viral RNAs themselves are responsible for guiding this process.…”

Section: How Do Rna Structures Direct Viral Rna Replication?mentioning

confidence: 99%

The myriad roles of RNA structure in the flavivirus life cycle

Abram,

Landry,

Wang

et al. 2024

RNA Biology

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As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

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Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly

Rheault

Cousineau

Fox

et al. 2023

Nucleic Acids Research

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Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or ‘riboswitch’, allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5′ untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.

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

Cited by 3 publications

References 58 publications

Zika virus NS3 and NS5 proteins determine strain-dependent differences in dsRNA accumulation in a host cell type-dependent manner

Zika virus NS3 and NS5 proteins determine strain-dependent differences in dsRNA accumulation in a host cell type-dependent manner

The myriad roles of RNA structure in the flavivirus life cycle

Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly

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