Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides

Oh, Hyung Suk; Banerjee, Sravani; Aponte-Diaz, David; Sharma, Suresh D.; Aligo, Jason; Lodeiro, María F.; Ning, Gang; Sharma, Rajni; Arnold, Jamie J.; Cameron, Craig Ε.

doi:10.1371/journal.ppat.1007036

Cited by 21 publications

(28 citation statements)

References 61 publications

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“…We compared the changes to Arf1, GBF1, and PI4KB localization caused by 3CD to corresponding changes caused by infection. Here we took advantage of our observation that infection in the presence of the replication inhibitor, guanidine hydrochloride (GuHCl), is sufficient to induce to PI4P and membrane biogenesis [ 42 ]. Because viral proteins do not accumulate to a detectable level by the indirect immunofluorescence assay in the presence of GuHCl (anti-3D in column marked PV in Fig 5A ), we used relocalization of PI4P to distinguish uninfected cells from infected cells (compare anti-PI4P in column marked Mock to column marked PV in Fig 5A and 5B ).…”

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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“…This difference among viruses might reflect the different membrane origins of their replication organelles. 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].…”

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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“…One possible explanation for this outcome is that the RdRp is encoded by the 3D region of the viral genome and this region is also a component of the PV 3CD protein. The 3CD protein has well established roles in aspects of the lifecycle before, during, and after replication, including virion morphogenesis [29,42,50,51]. Observations such as these highlight the additional level of complexity associated with establishing a cause-and-effect relationship between biochemical properties of the RdRp and fitness, virulence, and/or pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

“…Thereafter, growth of each virus was far more distinct than observed for the replicon, with PS-KH PV much slower than IF-KH PV and IF-PS-KH PV was substantially faster than both double mutants ( Figure 2A). The differences in outcome could reflect the differences in replication efficiency and/or a direct consequence of the substitutions in 3D-coding sequence on virus assembly or spread caused by changes to 3CD protein or 3D-containing precursor protein [29,42]. After a productive infection, the exponential phase of genome replication by PV typically occurs between 2 and 6 h post-infection, with virus assembly occurring between 4 and 8 h post-infection.…”

Section: Characterization Of the Double-and Triple-mutant Viruses Andmentioning

confidence: 99%

RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Kim

Ellis

Woodman

et al. 2019

Genes

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Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.broad-spectrum therapeutics [2-4] and a target for function/mechanism-based strategies for viral attenuation [5-10]. One function/mechanism of the RdRp that has been targeted most is that required for faithful incorporation of nucleotides [5][6][7][8][9][10]. Changing RdRp fidelity decreases or increases the genetic variation of the viral population, which, in turn, decreases fitness and virulence of the viral population [11][12][13][14][15][16].Known RdRp variants exhibiting a high-fidelity phenotype can also exhibit a reduced speed of nucleotide incorporation, at least at the biochemical level [17]. Replication speed is also a determinant of viral fitness and virulence [18]. So, is it the increased fidelity or decreased speed of nucleotide addition that gives rise to the attenuated phenotype? Further complicating the fidelity-versus-speed question are the recent observations that changes to fidelity also have consequence for the efficiency of recombination [19][20][21][22][23][24]. Increased RdRp fidelity decreases recombination efficiency and vice versa [19][20][21][22][23][24]. It will likely be impossible to attribute a single biochemical property of the RdRp to biological outcome.The most extensively studied PV fidelity mutants encode RdRps with amino acid substitutions at sites remote from the active site. Constructing equivalent substitutions conferring equivalent phenotypes in RdRps other than PV is difficult if not impossible. Our laboratory has therefore pursued active-site-based strategies to manipulate the fidelity, speed, and/or recombination efficiency of the RdRp [25]. We have shown that a lysine (Lys-359 in PV) present in ...

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Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides

Cited by 21 publications

References 61 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

RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

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