In Situ Spatial Organization of Potato Virus A Coat Protein Subunits as Assessed by Tritium Bombardment

Baratova, Ludmila A.; Ефимов, А. В.; Добров, Е. Н.; Fedorova, Natalija V.; Hunt, Reet; Badun, G. A.; Ksenofontov, Alexander L.; Torrance, L.; Järvekülg, Lilian

doi:10.1128/jvi.75.20.9696-9702.2001

Cited by 63 publications

(61 citation statements)

References 33 publications

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“…To understand the mechanism behind such regulation, structural data describing the changes introduced by CK2 phosphorylation into the nucleic acid binding domains of the corresponding proteins are required. Recently, a structural model of PVA CP was proposed on the basis of the experimental data obtained by tritium bombardment combined with theoretical predictions of protein topology ( Baratova et al, 2001). According to this model, the CK2 phosphorylation site Thr-242 is located in an interconnecting loop immediately after the ␤7 strand (amino acids 235 to 241) inside the structural unit commonly found in RNA binding proteins.…”

Section: Discussionmentioning

confidence: 99%

“…The overall secondary structure of this mutant was compared with that of the wild-type PVA CP us- (A) Scheme of PVA CP and its substitution mutants. The surface-exposed N-and C-terminal domains of the protein (black boxes) are shown together with a predicted abCd structural motif (Baratova et al, 2001) commonly found in RNA binding proteins. The known functions attributed to different regions of the protein (Dolja et al, 1995;Mahajan et al, 1996;Fernández-Fernández et al, 2002) are given below the scheme.…”

Section: Structural Comparison Of Wild-type Pva Cp and Its Ck2 Phosphmentioning

confidence: 99%

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Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection [W]

et al. 2003

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We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The ␣ -catalytic subunit of CK2 (CK2 ␣ ) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatographytandem mass spectrometry. The tobacco CK2 ␣ gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2 ␣ in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and longdistance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.

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Section: Discussionmentioning

confidence: 99%

Section: Structural Comparison Of Wild-type Pva Cp and Its Ck2 Phosphmentioning

confidence: 99%

Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection [W]

et al. 2003

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“…Most experiments were carried out using the G7 strain, but fiber diffraction experiments used both strains, and because of the better orientation and higher quality of the diffraction data, data from the G6 strain were used in the analysis. The coat proteins of the two strains differ at only 3 amino acid residues (33), and the substitutions are all conservative and within 20 residues of the N terminus, known to be located at the outer surface of the virion and probably disordered (5,56). PVX was purified and stored as previously described (52).…”

Section: Methodsmentioning

confidence: 99%

“…Fourier transform infrared spectroscopy studies of PVX suggest that the virion surface is highly hydrated and that the bound water molecules help to maintain the surface structure of the virion (6). Spectroscopic studies show that coat proteins of potexviruses and potyviruses have similarly high ␣-helical contents (5,31).…”

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Structure of Flexible Filamentous Plant Viruses

Kendall

McDonald

Bian

et al. 2008

J Virol

100

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Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.Flexible filamentous plant viruses include at least 19 recognized genera (22), almost all in three families of singlestranded, positive-sense RNA viruses, the Potyviridae, the Flexiviridae, and the Closteroviridae. Members of the family Potyviridae account for almost a third of the total known plant virus species (22) and are responsible for more than half the viral crop damage in the world (37), infecting most economically important crops (32). Members of the family Flexiviridae (2), and particularly of the large genus Potexvirus, are also of considerable significance to agriculture (42). Both families show great potential for biotechnological applications, including protein expression and vaccine production (12, 54). Despite their importance, however, little is known about the structures of any of the flexible filamentous plant viruses, in sharp contrast to the amount of data on the rigid tobamoviruses (48,63) or the icosahedral plant viruses (15); flexibility, instability, and in many cases low levels of expression have made these viruses particularly intractable to structural studies. Structural and evolutionary relationships among the flexible filamentous plant viruses have been suggested (18,47,56,60), but there is very little sequence homology between the coat proteins of viruses in the different families, and there has hitherto been no structural support for such relationships at the level of either viral symmetry or coat protein folding. Indeed, reports of viral symmetry until now appeared to contradict hypotheses of evolutionary relationships.Soybean mosaic virus (SMV) is a potyvirus, that is, a member of the genus Potyvirus, the largest genus in the family Potyviridae (3). SMV is a major pathogen of soybeans, transmitted efficiently through seed and by aphids in a nonpersistent manner; yield losses as high as 35% have been reported (30). Despite dramatic morphological differences, members of the family resemble the icosahedral plant comoviruses and animal picornaviruses in genomic organization and replication strategy (32). Early electron microscopic observations found the potyviruses to be about 7,500 Å long and 120 Å in diameter, with helical pitches of about 34 Å (44, 60). A fiber diffraction study (51) of the tritimovirus wheat streak mosaic virus (WSMV) suggested that WSMV has 6.9 subunits per turn of the viral helix, but there was consider...

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“…This was based on the finding that phosphorylation of Potato virus A (PVA; genus Potyvirus) CP downregulates its RNA binding function (11). The phosphorylation site within PVA CP was found to localize to a predicted abCd structural motif (12) common to RNA binding proteins. In addition, a chaperone-mediated ubiquitin degradation pathway has been suggested to limit the amount of CP during the early stages of infection (9).…”

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confidence: 99%

Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

Besong-Ndika

Ivanov

Hafrén

et al. 2015

J Virol

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Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCEThe main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production. P lant viral coat proteins (CPs) are associated with surprisingly many functions during the infectious cycle. Besides their roles in encapsidation and movement, CPs are implicated in viral RNA translation and replication during the early stages of infection (see references 1, 2, and 3 for reviews). Some of these functions involve specific and nonspecific interactions with viral RNA (4-7). For several positive-sense RNA viruses, CP modulates translation and/or replication in a concentration-dependent manner (4, 5). In these viruses, higher CP concentrations repress RNA accumulation, whereas lower concentrations stimulate RNA accumulation and translation. Hence, depending on the amount of CP, the virus can regulate the progression of virus infection from genome replication and translation to virion assembly.The potyviral CP is produced together with repl...

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In Situ Spatial Organization of Potato Virus A Coat Protein Subunits as Assessed by Tritium Bombardment

Cited by 63 publications

References 33 publications

Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection [W]

Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection [W]

Structure of Flexible Filamentous Plant Viruses

Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

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