The Norovirus NS3 Protein Is a Dynamic Lipid- and Microtubule-Associated Protein Involved in Viral RNA Replication

Cotton, Ben T; Hyde, Jennifer; Sarvestani, Soroush T.; Sosnovtsev, Stanislav V.; Green, Kim Y.; White, Peter A.; Mackenzie, Jason M.

doi:10.1128/jvi.02138-16

Cited by 32 publications

(43 citation statements)

References 38 publications

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“…For noroviruses, it has been found that their vRNAs contain multiple cis-acting elements and RNA secondary structures, which are important for norovirus replication (13,(51)(52)(53). With that in mind, it is intriguing that we have observed the ability of NS3 to enhance NS7/RdRP-mediated noroviral RNA synthesis in vitro, particularly since norovirus NS3 has recently been found to associate with vRI-dsRNAs in vRC on intracellular membranes (34)(35)(36). These findings indicate the critical roles of NS3 in norovirus RNA replication.…”

Section: Discussionmentioning

confidence: 95%

“…Consistent with this observation, the NS3 protein encoded by Southampton virus (SHV), a GI HuNoV, has been shown to have NTPase activity (33). In addition, norovirus NS3 has been recently reported to associate with vRI-dsRNAs in a viral replication complex (vRC) and to induce vesicular structures (34)(35)(36). However, previous work has yet to demonstrate RNA helicase or chaperoning activity associated with NS3 or any other norovirus-or calicivirus-encoded proteins or that the replication of norovirus or calicivirus requires the participation of any viral or host RNA-remodeling activities.…”

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confidence: 77%

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Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Hosmillo

Schwanke

et al. 2018

J Virol

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RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by and highlight the functional significance of NS3 in the noroviral life cycle. Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

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Section: Discussionmentioning

confidence: 95%

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confidence: 77%

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Hosmillo

Schwanke

et al. 2018

J Virol

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“…For RNA viruses, antibodies specific for double-stranded RNA, an intermediate formed during RNA replication, are also used to label active VRCs. Anti-dsRNA antibodies work particularly well for positive-sense RNA viruses (Cotton et al, 2017;Fontana et al, 2007;Welsch et al, 2009) and some negative-sense RNA viruses (Fontana et al, 2008;Mateer et al, 2019). For bunyaviruses and flaviviruses it has been demonstrated that the anti-dsRNA signal co-localizes with that of BrU incorporation sites (Fontana et al, 2008;Westaway et al, 1999).…”

Section: Immunogold Labellingmentioning

confidence: 99%

The viral replication organelles within cells studied by electron microscopy

Sachse

Castro

Tenorio

et al. 2019

Advances in Virus Research

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Transmission electron microscopy (TEM) has been crucial to study viral infections. As a result of recent advances in light and electron microscopy, we are starting to be aware of the variety of structures that viruses assemble inside cells. Viruses often remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. Here we revise the most relevant imaging technologies to study the biogenesis of viral replication organelles. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ in two and three dimensions are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral life cycle events with a detail never seen before.

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“…It was recently demonstrated that HuNoV NS3 had both ATP-dependent RNA helicase activity and ATP-independent RNA-chaperoning activity [26], while we were preparing our MNV NS3 manuscript. This study reports that MNV NS3, associated with the viral replication complex [27], has an NTPase activity as well as an RNA chaperone activity to destabilize dsRNA in the presence of Mg 2+ or Mn 2+ in fluorescence resonance energy transfer (FRET)-based assays.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3

Han

Lee

Kotiguda

et al. 2018

Journal of General Virology

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Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra-or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg 2+ -dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg 2+ or Mn 2+ in an NTPindependent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRETbased RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

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The Norovirus NS3 Protein Is a Dynamic Lipid- and Microtubule-Associated Protein Involved in Viral RNA Replication

Cited by 32 publications

References 38 publications

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

The viral replication organelles within cells studied by electron microscopy

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3

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