The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M.; Li, Zhenlu; Gohara, David W.; Buck, Matthias; Cremer, Paul S.; Boehr, David D.; Cameron, Craig Ε.

doi:10.1016/j.str.2017.11.001

Cited by 23 publications

(34 citation statements)

References 63 publications

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“…A fluorescence probe in the bilayer senses protein binding, causing fluorescence quenching for the proteins used to date. The system was validated using the Pleckstrin homology (PH) domain from phospholipase C δ1 that binds to PIP2 [ 47 ] and has now been validated for use with PI4P as well [ 48 ].…”

Section: Resultsmentioning

confidence: 99%

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

et al. 2018

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RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

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Section: Resultsmentioning

confidence: 99%

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

et al. 2018

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“…For example, poliovirus (PV) and coxsackievirus B3 hijack the Golgi and TGN and recruit PI4K to the replication organelle to produce PI(4)P in situ [67,75,76]. The 3C replication protein binds directly to PI(4)P and PI(4)P is required for viral RNA synthesis [75,77]. PI(4)P is also required for sterol enrichment within the replication organelles of Rhinovirus and PV [78,79].…”

Section: Discussionmentioning

confidence: 99%

Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus

Feng

Kovalev

et al. 2019

PLoS Pathog

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Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

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“…While the other functions that the HuNoV protease plays in the viral life cycle have yet to be fully elucidated, previous work has demonstrated that the HuNoV protease has RNA binding activity ( 32 ), as well as the ability to cleave cellular protease substrates ( 33 ). Work on the closely related poliovirus protease has also identified a phosphoinositide binding domain ( 34 ), but it remains to be determined whether the A105V mutation affects either of these or other as yet to be identified functions. It is noteworthy that the A105V mutation was not detected in isolation and was found only in combination with I109V.…”

Section: Discussionmentioning

confidence: 99%

Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro

Kitano

Hosmillo

Emmott

et al. 2018

Antimicrob Agents Chemother

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Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro. Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.

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The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain

Cited by 23 publications

References 63 publications

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus

Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro

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