Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Wang, Zhaowei; Qiu, Yang; Liu, Yongxiang; Qi, Nan; Si, Jianmin; Xia, Xiaoling; Wu, Di; Hu, Yuanyang; Zhou, Xi

doi:10.1074/jbc.m113.492728

Cited by 19 publications

(29 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2B). The Mn 2ϩ value is in agreement with previous biochemical characterizations (27,28), while the Mg 2ϩ concentration was similar to the amount previously used in cell extracts (26). Specific radioactivity (counts per minute per nanogram of RNA) of total RNA generated in the presence of 18 mM Mg 2ϩ or 1 mM Mn 2ϩ ( Fig.…”

Section: Resultssupporting

confidence: 89%

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Short

Speir

Gopal

et al. 2016

J Virol

View full text Add to dashboard Cite

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ϳ50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single-and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

show abstract

Section: Resultssupporting

confidence: 89%

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Short

Speir

Gopal

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…To solve this issue, we have designed a set of experiments in our previous study [41], which could help determine if the TNTase activity of FHV protein A can function to restore the replicability of template with 3′-terminal nucleotide(s) loss.…”

Section: Resultsmentioning

confidence: 99%

“…The different sensitivity to RdRP inhibitors is possibly due to the de novo and polyadenylation-independent characteristic of nodaviral or noroviral RdRP [50]. Besides, the amino acid sequences of nodaviral RdRPs are partly similar to DNA polymerase by sequence analyses [41], which may be a cause for the inhibitor selectivity. Based on this and previous studies of FHV and WhNV, the de novo initiation may be a general mechanism in the family Nodaviridae .…”

Section: Discussionmentioning

confidence: 99%

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Wang

Xia

et al. 2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.

show abstract

“…WhNV Protein A GAA is provided by the plasmid pA GAA . The RdRp activity of Protein A GAA is nulled by mutating the GDD replication site to GAA as previously described Wang et al, 2013). Protein A GAA retained its RNA1 recruitment activity despite losing its RNA replicase activity .…”

Section: Resultsmentioning

confidence: 93%

The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

et al. 2014

Self Cite

View full text Add to dashboard Cite

RNA replication of positive-strand (+)RNA viruses requires the lipids present in intracellular membranes, the sites of which viral replicases associate with. However, the direct effects of membrane lipids on viral replicases are still poorly understood. Wuhan nodavirus (WhNV) protein A, which associates with mitochondrial membranes, is the sole replicase required for RNA replication. Here, we report that WhNV protein A binds to RNA1 in a cooperative manner. Moreover, mitochondrial membrane lipids (MMLs) stimulated the RNA binding activity and cooperativity of protein A, and such stimulations exhibited strong selectivity for distinct phospholipids. Interestingly, MMLs stimulated the RNA-binding cooperativity only at higher protein A concentrations. Further investigation showed that MMLs stimulate the RNA binding of protein A by promoting its self-interaction. Finally, manipulating MML metabolism affected the protein A-induced RNA1 recruitment in cells. Together, our findings reveal the direct effects of membrane lipids on the RNA binding activity of a nodaviral replicase.

show abstract

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Cited by 19 publications

References 74 publications

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

Contact Info

Product

Resources

About