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Kristiina Mäkinen - One of the best experts on this subject based on the ideXlab platform.

  • dynAmics of protein AccumulAtion from the 3 end of virAl rnA Are different from those in the rest of the genome in PotAto Virus A infection
    Journal of Virology, 2019
    Co-Authors: Shreya Saha, Anders Hafrén, Kristiina Mäkinen
    Abstract:

    One lArge open reAding frAme (ORF) encodes 10 potyvirAl proteins. We compAred the AccumulAtion of cylindricAl inclusion (CI) protein from the middle, coAt protein (CP) from the 3′end, And RenillA luciferAse (RLUC) from two distinct locAtions in PotAto Virus A (PVA) RNA. 5′ RLUC wAs expressed from An rluc gene inserted between the P1 And helper component proteinAse (HCPro) cistrons, And 3′ RLUC wAs expressed from the gene inserted between the RNA polymerAse And CP cistrons. VirAl protein And RNA AccumulAtion were quAntitAted (i) when expressed from PVA RNA in the presence of ectopicAlly expressed genome-linked virAl protein (VPg) And AuxiliAry proteins And (ii) At different time points during nAturAl infection. The rAte And timing of 3′ RLUC And CP AccumulAtion were found to be different from those of 5′ RLUC And CI. Ectopic expression of VPg boosted PVA RNA, 3′ RLUC, And, together with HCPro, CP AccumulAtion, whereAs 5′ RLUC And CI AccumulAtion remAined unAffected regArdless of the increAsed virAl RNA Amount. In nAturAl infection, the rAte of the noteworthy minute eArly AccumulAtion of 3′ RLUC AccelerAted towArd the end of infection. 5′ RLUC AccumulAtion, which wAs AlreAdy pronounced At 2 dAys postinfection, increAsed moderAtely And stAbilized to A constAnt level by dAy 5, whereAs PVA RNA And CP levels continued to increAse throughout the infection. We propose thAt these observAtions connect with the mechAnisms by which potyVirus infection limits CP AccumulAtion during eArly infection And specificAlly supports its AccumulAtion lAte in infection, but follow-up studies Are required to understAnd the mechAnism of how this occurs. IMPORTANCE The results of this study suggest thAt the dynAmics of potyvirAl protein AccumulAtion Are regulAted differentiAlly from the 3′ end of virAl RNA thAn from the rest of the genome, the significAnce of which would be to sAtisfy the needs of replicAtion eArly And pArticle Assembly lAte in infection.

  • coAt protein regulAtion by ck2 cpip hsp70 And chip is required for PotAto Virus A replicAtion And coAt protein AccumulAtion
    Journal of Virology, 2017
    Co-Authors: Andres Lohmus, Anders Hafrén, Kristiina Mäkinen
    Abstract:

    We demonstrAte here thAt both coAt protein (CP) phosphorylAtion by protein kinAse CK2 And A chAperone system formed by two heAt shock proteins, CP-interActing protein (CPIP) And heAt shock protein 70 (HSP70), Are essentiAl for PotAto Virus A (PVA; genus PotyVirus) replicAtion And thAt All these host proteins hAve the cApAcity to contribute to the level of PVA CP AccumulAtion. An E3 ubiquitin ligAse cAlled cArboxyl terminus Hsc70-interActing protein (CHIP), which mAy pArticipAte in the CPIP-HSP70-mediAted CP degrAdAtion, is Also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP wAs found to be essentiAl for virAl replicAtion And to regulAte CP protein stAbility. Substitution of Thr243 either with A phosphorylAtion-mimicking Asp (CPADA) or with A phosphorylAtion-deficient AlA (CPAAA) residue in CP expressed from virAl RNA limited PVA gene expression to the level of nonreplicAting PVA. We found thAt both the CPAAA mutAnt And CK2 silencing inhibited, whereAs CPADA mutAnt And overexpression of CK2 increAsed, PVA trAnslAtion. From our previous studies, we know thAt phosphorylAtion reduces the RNA binding cApAcity of PVA CP And An excess of CP fully blocks virAl RNA trAnslAtion. Together, these findings suggest thAt binding by nonphosphorylAted PVA CP represses virAl RNA trAnslAtion, involving further CP phosphorylAtion And CPIP-HSP70 chAperone Activities As prerequisites for PVA replicAtion. We propose thAt this mechAnism contributes to shifting potyVirus RNA from trAnslAtion to replicAtion. ImportAnce Host protein kinAse CK2, two host chAperones, CPIP And HSP70, And virAl coAt protein (CP) phosphorylAtion At Thr243 Are needed for PotAto Virus A (PVA) replicAtion. Our results show thAt nonphosphorylAted CP blocks virAl trAnslAtion, likely viA binding to virAl RNA. We propose thAt this trAnslAtionAl block is needed to Allow time And spAce for the formAtion of potyvirAl replicAtion complex Around the 3' end of virAl RNA. Progression into replicAtion involves CP regulAtion by both CK2 phosphorylAtion And chAperones CPIP And HSP70.

  • protein composition of 6k2 induced membrAne structures formed during PotAto Virus A infection
    Molecular Plant Pathology, 2016
    Co-Authors: Andres Lohmus, Markku Varjosalo, Kristiina Mäkinen
    Abstract:

    The definition of the precise moleculAr composition of membrAnous replicAtion compArtments is A key to understAnding the mechAnisms of Virus multiplicAtion. Here, we set out to investigAte the protein composition of the potyvirAl replicAtion complexes. We purified the potyvirAl 6K2 protein-induced membrAnous structures from PotAto Virus A (PVA)-infected NicotiAnA benthAmiAnA plAnts. For this purpose, the 6K2 protein, which is the mAin inducer of potyvirAl membrAne reArrAngements, wAs expressed in fusion with An N-terminAl Twin-Strep-tAg And CeruleAn fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tAgged CeruleAn-6K2 (C6K) Virus And the SC6K protein Alone in the Absence of infection were used As controls. A purificAtion scheme exploiting discontinuous sucrose grAdient centrifugAtion followed by Strep-tAg-bAsed Affinity chromAtogrAphy wAs developed. Both (+)- And (-)-strAnd PVA RNA And virAl protein VPg were co-purified specificAlly with the Affinity tAgged PVA-SC6K. The purified sAmples, which contAined individuAl vesicles And membrAne clusters, were subjected to mAss spectrometry AnAlysis. DAtA AnAlysis reveAled thAt mAny of the detected virAl And host proteins were either significAntly enriched or fully specificAlly present in PVA-SC6K sAmples when compAred with the controls. Eight of eleven potyvirAl proteins were identified with high confidence from the purified membrAne structures formed during PVA infection. RibosomAl proteins were identified from the 6K2-induced membrAnes only in the presence of A replicAting Virus, reinforcing the tight coupling between replicAtion And trAnslAtion. A substAntiAl number of proteins AssociAting with chloroplAsts And severAl host proteins previously linked with potyVirus replicAtion complexes were co-purified with PVA-derived SC6K, supporting the conclusion thAt the host proteins identified in this study mAy hAve relevAnce in PVA replicAtion.

  • TowArd the Reconstitution of A Two-Enzyme CAscAde for ResverAtrol Synthesis on PotyVirus PArticles
    Frontiers in Plant Science, 2016
    Co-Authors: Jane Besong-ndika, Thierry Michon, Jan Pille, Jocelyne Walter, Matti Wahlsten, Daniela Cardinale, Kristiina Mäkinen
    Abstract:

    The highly ordered protein bAckbone of Virus pArticles mAkes them AttrActive cAndidAtes for use As enzyme nAno-cArriers (ENCs). We hAve previously developed A non-covAlent And versAtile ApproAch for Adhesion of enzymes to Virus pArticles. This ApproAch mAkes use of z33, A peptide derived from the B-domAin of StAphylococcus Aureus protein A, which binds to the Fc domAin of mAny immunoglobulins. We hAve demonstrAted thAt with specific Antibodies Addressed AgAinst the virAl cApsid proteins (CPs) An 87% coverAge of z33-tAgged proteins cAn be Achieved on potyVirus pArticles. 4-coumArAte coenzyme A ligAse (4CL2) And stilbene synthAse (STS) cAtAlyze consecutive steps in the resverAtrol synthetic pAthwAy. In this study, these enzymes were modified to cArry An N-terminAl z33 peptide And A C-terminAl 6xHis tAg to obtAin z4CL2His And zSTSHis, respectively. A protein chimerA, z4CL2::STSHis, with the sAme modificAtions wAs Also generAted from the genetic fusion of both mono-enzyme encoding genes. All z33 enzymes were biologicAlly Active After expression in EscherichiA coli As reveAled by LC-MS AnAlysis to identify resverAtrol And Assembled reAdily into mAcromoleculAr complexes with PotAto Virus A pArticles And α-PVA CP Antibodies. To test simultAneous immobilizAtion-purificAtion, we Applied the double Antibody sAndwich – ELISA protocol to cApture Active z33-contAing mono-enzymes And protein chimerA directly from clArified soluble cell lysAtes onto the Virus pArticle surfAce. These immobilized enzymes were Able to synthesize resverAtrol. We present here A bottom up ApproAch to immobilize Active enzymes onto Virus-bAsed ENCs And discuss the potentiAl to utilize this method in the purificAtion And configurAtion of nAno-devices.

  • formAtion of PotAto Virus A induced rnA grAnules And virAl trAnslAtion Are interrelAted processes required for optimAl Virus AccumulAtion
    PLOS Pathogens, 2015
    Co-Authors: Anders Hafrén, Andres Lohmus, Kristiina Mäkinen
    Abstract:

    RNA grAnules Are cellulAr structures, which plAy An importAnt role in mRNA trAnslAtion, storAge, And degrAdAtion. AnimAl (+)RNA Viruses often co-opt RNA grAnule proteins for virAl reproduction. However, the role of RNA grAnules in plAnt virAl infections is poorly understood. Here we use PotAto Virus A (PVA) As A model potyVirus And demonstrAte thAt the helper component-proteinAse (HCpro), the potyvirAl suppressor of RNA silencing, induces the formAtion of RNA grAnules. We used confocAl microscopy to demonstrAte the presence of host RNA binding proteins including Acidic ribosomAl protein P0, ArgonAute 1 (AGO1), oligouridylAte-binding protein 1 (UBP1), vAricose (VCS) And eukAryotic initiAtion fActor iso4E (eIF(iso)4E) in these potyVirus-induced RNA grAnules. We show thAt the number of potyvirAl RNA grAnules is down-regulAted by the genome-linked virAl protein (VPg). We demonstrAted previously thAt VPg is A Virus-specific trAnslAtionAl regulAtor thAt co-operAtes with potyvirAl RNA grAnule components P0 And eIF(iso)4E in PVA trAnslAtion. In this study we show thAt HCpro And vAricose, components of potyvirAl RNA grAnules, stimulAte VPg-promoted trAnslAtion of the PVA, whereAs UBP1 inhibits this process. Hence, we propose thAt PVA trAnslAtion operAtes viA A pAthwAy thAt is interrelAted with potyvirAl RNA grAnules in PVA infection. The importAnce of these grAnules is evident from the strong reduction in virAl RNA And coAt protein Amounts thAt follows knock down of potyvirAl RNA grAnule components. HCpro suppresses AntivirAl RNA silencing during infection, And our results Allow us to propose thAt this is Also the functionAl context of the potyvirAl RNA grAnules we describe in this study.

Jari P. T. Valkonen - One of the best experts on this subject based on the ideXlab platform.

  • A novel interAction network used by potyViruses in Virus host interActions At the protein level
    Viruses, 2019
    Co-Authors: Marjo Alapoikela, Minna-liisa Rajamäki, Jari P. T. Valkonen
    Abstract:

    Host proteins thAt Are centrAl to infection of potyViruses (genus PotyVirus; fAmily PotyviridAe) include the eukAryotic trAnslAtion initiAtion fActors eIF4E And eIF(iso)4E. The potyvirAl genome-linked protein (VPg) And the helper component proteinAse (HCpro) interAct with eAch other And with eIF4E And eIF(iso)4E And proteins Are involved in the sAme functions during virAl infection. VPg interActs with eIF4E/eIF(iso)4E viA the 7-methylguAnosine cAp-binding region, whereAs HCpro interActs with eIF4E/eIF(iso)4E viA the 4E-binding motif YXXXXLΦ, similAr to the motif in eIF4G. In this study, HCpro And VPg were found to interAct in the nucleus, nucleolus, And cytoplAsm in cells infected with the potyVirus PotAto Virus A (PVA). In the cytoplAsm, interActions between HCpro And VPg occurred in punctAte bodies not AssociAted with virAl replicAtion vesicles. In Addition to HCpro, the 4E-binding motif wAs recognized in VPg of PVA. MutAtions in the 4E-binding motif of VPg from PVA weAkened interActions with eIF4E And heAvily reduced PVA virulence. Furthermore, mutAtions in the 4G-binding domAin of eIF4E reduced interActions with VPg And Abolished interActions with HCpro. Thus, HCpro And VPg cAn both interAct with eIF4E using the 4E-binding motif. Our results suggest A novel interAction network used by potyViruses to interAct with host plAnts viA trAnslAtion initiAtion fActors.

  • mutAtion of A short vAriAble region in hcpro protein of PotAto Virus A Affects interActions with A microtubule AssociAted protein And induces necrotic responses in tobAcco
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Tuuli Haikonen, Minna-liisa Rajamäki, Yanping Tian, Jari P. T. Valkonen
    Abstract:

    Helper component proteinAse (HCpro) is A multifunctionAl protein of potyViruses (genus PotyVirus). HCpro of PotAto Virus A (PVA) interActs with the microtubule-AssociAted protein HIP2 in host cells, And depletion of HIP2 reduces Virus AccumulAtion. This study shows thAt HCpro of PotAto Virus Y And TobAcco etch Virus Also interAct with HIP2. The C-proximAl portion of PVA HCpro determines the interAction with HIP2 And wAs found to contAin A stretch of six residues comprising A highly vAriAble region (HVR) in potyViruses. MutAtions in HVR reduced PVA AccumulAtion in tobAcco plAnts And induced necrotic symptoms novel to PVA. MicroArrAy And quAntitAtive reverse trAnscription polymerAse chAin reAction AnAlyses reveAled induction of mAny defense-relAted genes including ethylene- And jAsmonic Acid–inducible pAthwAys in systemicAlly infected leAves At necrosis onset. SAlicylic Acid–mediAted signAling wAs dispensAble for the response. Genes relAted to microtubule functions were down-regulAted. StructurAl modeling o...

  • interAction of the microtubule AssociAted host protein hip2 with virAl helper component proteinAse is importAnt in infection with PotAto Virus A
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Tuuli Haikonen, Minna-liisa Rajamäki, Jari P. T. Valkonen
    Abstract:

    Microtubules (MT) outline And mAintAin the overAll shApe of cells And cAn reorgAnize cellulAr membrAnes to serve As sites of RNA Virus replicAtion. Here, we provide dAtA on involvement of An MT-AssociAted protein in infection of plAnts with A potyVirus, PotAto Virus A (PVA), representing the lArgest fAmily of plAnt-infecting RNA Viruses. Our results showed thAt helper-component proteinAse (HCpro)-interActing protein 2 (HIP2) of PotAto (SolAnum tuberosum) is An MT-AssociAted protein similAr to ArAbidopsis SPR2. Virus-induced silencing of HIP2 in NicotiAnA benthAmiAnA resulted in A spirAl-like growth phenotype, similAr to the ArAbidopsis spr2 mutAnt, And the spr2 phenotype in ArAbidopsis wAs complemented with PotAto HIP2. HCpro of PVA interActed with HIP2 of PotAto And tobAcco (NicotiAnA tAbAcum). The interAction wAs detected by bimoleculAr fluorescence complementAtion in PVA-infected leAves on MT And MT intersections At the cell cortex. HIP2-HCpro interAction wAs determined by the C-proximAl α-helix-rich d...

  • helper component proteinAse of the genus potyVirus is An interAction pArtner of trAnslAtion initiAtion fActors eif iso 4e And eif4e And contAins A 4e binding motif
    Journal of Virology, 2011
    Co-Authors: Marjo Alapoikela, Minna-liisa Rajamäki, Elisa Goytia, Tuuli Haikonen, Jari P. T. Valkonen
    Abstract:

    The multifunctionAl helper component proteinAse (HCpro) of potyViruses (genus PotyVirus; PotyviridAe) shows self-interAction And interActs with other potyvirAl And host plAnt proteins. Host proteins thAt Are pivotAl to potyVirus infection include the eukAryotic trAnslAtion initiAtion fActor eIF4E And the isoform eIF(iso)4E, which interAct with virAl genome-linked protein (VPg). Here we show thAt HCpro of PotAto Virus A (PVA) interActs with both eIF4E And eIF(iso)4E, with interActions with eIF(iso)4E being stronger, As judged by the dAtA of A yeAst two-hybrid system AssAy. A bimoleculAr fluorescence complementAtion AssAy on leAves of NicotiAnA benthAmiAnA showed thAt HCpro from three potyViruses (PVA, PotAto Virus Y, And TobAcco etch Virus) interActed with the eIF(iso)4E And eIF4E of tobAcco (NicotiAnA tAbAcum); interActions with eIF(iso)4E And eIF4E of PotAto (SolAnum tuberosum) were weAker. In PVA-infected cells, interActions between HCpro And tobAcco eIF(iso)4E were confined to round structures thAt colocAlized with 6K2-induced vesicles. Point mutAtions introduced to A 4E binding motif identified in the C-terminAl region of HCpro debilitAted interActions of HCpro with trAnslAtion initiAtion fActors And were detrimentAl to the virulence of PVA in plAnts. The 4E binding motif conserved in HCpro of potyViruses And HCpro-initiAtion fActor interActions suggest new roles for HCpro And/or trAnslAtion fActors in the potyVirus infection cycle.

  • Control of nucleAr And nucleolAr locAlizAtion of nucleAr inclusion protein A of picornA-like PotAto Virus A in NicotiAnA species.
    The Plant cell, 2009
    Co-Authors: Minna-liisa Rajamäki, Jari P. T. Valkonen
    Abstract:

    The multifunctionAl nucleAr inclusion protein A (NIA) of potyViruses (genus PotyVirus; PotyviridAe) AccumulAtes in the nucleus of Virus-infected cells for unknown reAsons. In this study, two regions in the virAl genome-linked protein (VPg) domAin of NIA in PotAto Virus A (PVA) were found to constitute nucleAr And nucleolAr locAlizAtion signAls (NLS) in plAnt cells (NicotiAnA spp). Amino Acid substitutions in both NLS I (residues 4 to 9) And NLS II (residues 41 to 50) prevented nucleAr locAlizAtion, whereAs mutAtions in either single NLS did not. MutAtions in either NLS, however, prevented nucleolAr locAlizAtion And prevented or diminished Virus replicAtion in protoplAsts, AccumulAtion in infected plAnt tissues, And/or systemic movement in plAnts. One NLS mutAnt wAs pArtiAlly complemented by the wild-type VPg expressed in trAnsgenic plAnts. Furthermore, NLS I controlled NIA AccumulAtion in CAjAl bodies. The VPg domAin interActed with fibrillArin, A nucleolAr protein, And depletion of fibrillArin reduced PVA AccumulAtion. Overexpression of VPg in leAf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereAs NLS I And NLS II mutAnts, which exhibited reduced nucleAr And nucleolAr locAlizAtion, showed no such Activity. These results demonstrAte thAt some of the most essentiAl virAl functions required for completion of the infection cycle Are tightly linked to regulAtion of the NIA nucleAr And nucleolAr locAlizAtion.

Minna-liisa Rajamäki - One of the best experts on this subject based on the ideXlab platform.

  • A novel interAction network used by potyViruses in Virus host interActions At the protein level
    Viruses, 2019
    Co-Authors: Marjo Alapoikela, Minna-liisa Rajamäki, Jari P. T. Valkonen
    Abstract:

    Host proteins thAt Are centrAl to infection of potyViruses (genus PotyVirus; fAmily PotyviridAe) include the eukAryotic trAnslAtion initiAtion fActors eIF4E And eIF(iso)4E. The potyvirAl genome-linked protein (VPg) And the helper component proteinAse (HCpro) interAct with eAch other And with eIF4E And eIF(iso)4E And proteins Are involved in the sAme functions during virAl infection. VPg interActs with eIF4E/eIF(iso)4E viA the 7-methylguAnosine cAp-binding region, whereAs HCpro interActs with eIF4E/eIF(iso)4E viA the 4E-binding motif YXXXXLΦ, similAr to the motif in eIF4G. In this study, HCpro And VPg were found to interAct in the nucleus, nucleolus, And cytoplAsm in cells infected with the potyVirus PotAto Virus A (PVA). In the cytoplAsm, interActions between HCpro And VPg occurred in punctAte bodies not AssociAted with virAl replicAtion vesicles. In Addition to HCpro, the 4E-binding motif wAs recognized in VPg of PVA. MutAtions in the 4E-binding motif of VPg from PVA weAkened interActions with eIF4E And heAvily reduced PVA virulence. Furthermore, mutAtions in the 4G-binding domAin of eIF4E reduced interActions with VPg And Abolished interActions with HCpro. Thus, HCpro And VPg cAn both interAct with eIF4E using the 4E-binding motif. Our results suggest A novel interAction network used by potyViruses to interAct with host plAnts viA trAnslAtion initiAtion fActors.

  • ClAss 1 RNAseIII endoribonucleAses of PPIV (PPR3) And SPCSV (CSR3) suppress RNAi in leAves of NicotiAnA benthAmiAnA.
    2015
    Co-Authors: Isabel Weinheimer, Minna-liisa Rajamäki, Mart Saarma, Yaming Jiu, Olli Matilainen, Jukka Kallijärvi, Wilmer J. Cuellar, Carina I. Holmberg, Jussi Jäntti
    Abstract:

    (A) The RNAseIII signAture motif (blue) And dsRNA-binding domAin (red) conserved in ClAss 1 RNAseIII endoribonucleAses, And substitution of two conserved Amino Acid residues of the cAtAlytic site Are depicted (mutAted proteins PPR3-AlA And CSR3-AlA). (B) Suppression of sense ssRNA—induced RNAi. The left And right side of leAves of gfp-trAnsgenic N. benthAmiAnA line 16c were infiltrAted with AgrobActerium tumefAciens (Agro) strAins expressing gfp to induce silencing of the constitutively expressed gfp trAnsgene (note green fluorescence in leAf veins) And co-infiltrAted with Agro strAins for expression of PPR3, PPR3-AlA, CSR3, CSR3-AlA, or GUS (β-glucuronidAse, negAtive control) or mock-infiltrAted with buffer (negAtive control). LeAves were photogrAphed And AnAlyzed 3 dAys post-infiltrAtion. AccumulAtion of gfp mRNA And siRNA wAs AnAlyzed by northern blotting. Ethidium bromide—stAined gels of rRNA were used As loAding controls. (C) Immunoblot of the RNAseIII proteins in the infiltrAted tissues shown in (B). Control indicAtes purified recombinAnt CSR3 (positive control). (D) Suppression of dsRNA (hAirpin RNA)-induced RNAi. Co-infiltrAtion wAs cArried out As in (B), except thAt An Agro strAin expressing double-strAnded (hAirpin) gfp wAs used. Agro strAins expressing GUS or the SPCSV p22 silencing suppressor were used As A negAtive And A positive control, respectively. AccumulAtion of gfp mRNA And siRNA wAs determined by northern blotting. (E) Reversion of RNAi. Suppression of the gfp trAnsgene wAs Achieved by sense-mediAted silencing As in (B). After 24 h, the sAme leAf spots were infiltrAted with An Agro strAin for expression of PPR3, PPR3-AlA, CSR3, CSR3-AlA, GUS (negAtive control), or the HCpro silencing suppressor of PotAto Virus A (positive control) And photogrAphed 3 dAys lAter.

  • interAction of the microtubule AssociAted host protein hip2 with virAl helper component proteinAse is importAnt in infection with PotAto Virus A
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Tuuli Haikonen, Minna-liisa Rajamäki, Jari P. T. Valkonen
    Abstract:

    Microtubules (MT) outline And mAintAin the overAll shApe of cells And cAn reorgAnize cellulAr membrAnes to serve As sites of RNA Virus replicAtion. Here, we provide dAtA on involvement of An MT-AssociAted protein in infection of plAnts with A potyVirus, PotAto Virus A (PVA), representing the lArgest fAmily of plAnt-infecting RNA Viruses. Our results showed thAt helper-component proteinAse (HCpro)-interActing protein 2 (HIP2) of PotAto (SolAnum tuberosum) is An MT-AssociAted protein similAr to ArAbidopsis SPR2. Virus-induced silencing of HIP2 in NicotiAnA benthAmiAnA resulted in A spirAl-like growth phenotype, similAr to the ArAbidopsis spr2 mutAnt, And the spr2 phenotype in ArAbidopsis wAs complemented with PotAto HIP2. HCpro of PVA interActed with HIP2 of PotAto And tobAcco (NicotiAnA tAbAcum). The interAction wAs detected by bimoleculAr fluorescence complementAtion in PVA-infected leAves on MT And MT intersections At the cell cortex. HIP2-HCpro interAction wAs determined by the C-proximAl α-helix-rich d...

  • mutAtion of A short vAriAble region in hcpro protein of PotAto Virus A Affects interActions with A microtubule AssociAted protein And induces necrotic responses in tobAcco
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Tuuli Haikonen, Minna-liisa Rajamäki, Yanping Tian, Jari P. T. Valkonen
    Abstract:

    Helper component proteinAse (HCpro) is A multifunctionAl protein of potyViruses (genus PotyVirus). HCpro of PotAto Virus A (PVA) interActs with the microtubule-AssociAted protein HIP2 in host cells, And depletion of HIP2 reduces Virus AccumulAtion. This study shows thAt HCpro of PotAto Virus Y And TobAcco etch Virus Also interAct with HIP2. The C-proximAl portion of PVA HCpro determines the interAction with HIP2 And wAs found to contAin A stretch of six residues comprising A highly vAriAble region (HVR) in potyViruses. MutAtions in HVR reduced PVA AccumulAtion in tobAcco plAnts And induced necrotic symptoms novel to PVA. MicroArrAy And quAntitAtive reverse trAnscription polymerAse chAin reAction AnAlyses reveAled induction of mAny defense-relAted genes including ethylene- And jAsmonic Acid–inducible pAthwAys in systemicAlly infected leAves At necrosis onset. SAlicylic Acid–mediAted signAling wAs dispensAble for the response. Genes relAted to microtubule functions were down-regulAted. StructurAl modeling o...

  • helper component proteinAse of the genus potyVirus is An interAction pArtner of trAnslAtion initiAtion fActors eif iso 4e And eif4e And contAins A 4e binding motif
    Journal of Virology, 2011
    Co-Authors: Marjo Alapoikela, Minna-liisa Rajamäki, Elisa Goytia, Tuuli Haikonen, Jari P. T. Valkonen
    Abstract:

    The multifunctionAl helper component proteinAse (HCpro) of potyViruses (genus PotyVirus; PotyviridAe) shows self-interAction And interActs with other potyvirAl And host plAnt proteins. Host proteins thAt Are pivotAl to potyVirus infection include the eukAryotic trAnslAtion initiAtion fActor eIF4E And the isoform eIF(iso)4E, which interAct with virAl genome-linked protein (VPg). Here we show thAt HCpro of PotAto Virus A (PVA) interActs with both eIF4E And eIF(iso)4E, with interActions with eIF(iso)4E being stronger, As judged by the dAtA of A yeAst two-hybrid system AssAy. A bimoleculAr fluorescence complementAtion AssAy on leAves of NicotiAnA benthAmiAnA showed thAt HCpro from three potyViruses (PVA, PotAto Virus Y, And TobAcco etch Virus) interActed with the eIF(iso)4E And eIF4E of tobAcco (NicotiAnA tAbAcum); interActions with eIF(iso)4E And eIF4E of PotAto (SolAnum tuberosum) were weAker. In PVA-infected cells, interActions between HCpro And tobAcco eIF(iso)4E were confined to round structures thAt colocAlized with 6K2-induced vesicles. Point mutAtions introduced to A 4E binding motif identified in the C-terminAl region of HCpro debilitAted interActions of HCpro with trAnslAtion initiAtion fActors And were detrimentAl to the virulence of PVA in plAnts. The 4E binding motif conserved in HCpro of potyViruses And HCpro-initiAtion fActor interActions suggest new roles for HCpro And/or trAnslAtion fActors in the potyVirus infection cycle.

Alexander L. Ksenofontov - One of the best experts on this subject based on the ideXlab platform.

  • the structure of the PotAto Virus A pArticles elucidAted by smAll Angle x rAy scAttering And complementAry techniques
    Biochemistry, 2021
    Co-Authors: E V Shtykova, Alexander L. Ksenofontov, Alexander M. Arutyunyan, Maxim V Petoukhov, N V Fedorova, E V Skurat, Larisa V Kordyukova, Andrey Moiseenko
    Abstract:

    PotAto Virus A (PVA) protein coAt contAins on its surfAce pArtiAlly unstructured N-terminAl domAin of the virAl coAt protein (CP), whose structurAl And functionAl chArActeristics Are importAnt for understAnding the mechAnism of plAnt infection with this Virus. In this work, we investigAted the properties And the structure of intAct PVA And pArtiAlly trypsinized PVAΔ32 virions using smAll-Angle X-rAy scAttering (SAXS) And complimentAry methods. It wAs shown thAt After the removAl of 32 N-terminAl Amino Acids of the CP, the virion did not disintegrAte And remAined compAct, but the helicAl pitch of the CP pAcking chAnged. To determine the nAture of these chAnges, we performed Ab initio modeling, including the multiphAse procedure, with the geometric bodies (helices) And restorAtion of the PVA structure in solution using AvAilAble high-resolution structures of the homologous CP from the PVY potyVirus, bAsed on the SAXS dAtA. As A result, for the first time, A low-resolution structure of the filAmentous PVA Virus, both intAct And pArtiAlly degrAded, wAs elucidAted under conditions close to nAturAl. The fAr-UV circulAr dichroism spectrA of the PVA And PVAΔ32 sAmples differed significAntly in the Amplitude And position of the mAin negAtive mAximum. The extent of thermAl denAturAtion of these sAmples in the temperAture rAnge of 20-55°C wAs Also different. The dAtA of trAnsmission electron microscopy showed thAt the PVAΔ32 virions were mostly rod-shAped, in contrAst to the flexible filAmentous pArticles typicAl of the intAct Virus, which correlAted well with the SAXS results. In generAl, structurAl AnAlysis indicAtes An importAnce of the CP N-terminAl domAin for the vitAl functions of PVA, which cAn be used to develop A strAtegy for combAting this plAnt pAthogen.

  • structure of PotAto Virus A coAt protein pArticles And their dissociAtion
    Molecular Biology, 2018
    Co-Authors: Alexander L. Ksenofontov, E. N. Dobrov, Alexander M. Arutyunyan, Natalia V. Fedorova, Lilian Järvekülg, A E Golanikov, E V Shtykova
    Abstract:

    This pAper reports on A complex structurAl AnAlysis of the PotAto Virus A coAt protein using A set of complementAry physico-chemicAl methods. We hAve demonstrAted previously thAt this protein does not exist As individuAl subunits in solution And undergoes AssociAtion into oligomers with subsequent trAnsition to β-conformAtion. The purpose of the present work wAs to study the possible mechAnisms of this trAnsformAtion And to seArch for methods thAt dissociAte protein oligomers. To AnAlyze the low resolution protein structure in solution, smAll-Angle X-rAy scAttering wAs used. StAble pArticles representing clusters of 30 coAt protein subunits were present even in An Aqueous sAlt solution with A high ionic strength And pH (pH 10.5; 0.5 M NACl). The pArticles did not dissociAte in the presence of 10 mM dextrAn sulfAtes (15 And 100 kDA). DissociAtion in the presence of 5.2 mM sodium dodecyl sulfAte results in the formAtion of the subunit-detergent complexes consisting of 10-12 smAll pArticles joined together like "beAds on A string". SimilAr effects of sodium dodecyl sulfAte were shown for serum Albumins (bovine And humAn). DenAturAtion of the PotAto Virus A coAt protein molecules occurs in the presence of detergent concentrAtions thAt Are seven times lower thAn thAt in Albumins (5.2 And 35 mM), which confirms low stAbility of the PotAto Virus A coAt protein. Using spectrAl methods, preservAtion of the secondAry structure And loss of the tertiAry structure of the protein in its complex with sodium dodecyl sulfAte hAve been demonstrAted. Possible mechAnism for protein pArticle formAtion through the interAction between unordered terminAl domAins And their trAnsformAtion into β-structures hAs been suggested.

  • PArtiAlly disordered structure in intrAVirus coAt protein of potyVirus PotAto Virus A.
    PloS one, 2013
    Co-Authors: Alexander L. Ksenofontov, Ludmila A. Baratova, Viiu Paalme, Alexander M. Arutyunyan, Pavel I. Semenyuk, Natalia V. Fedorova, Reet Rumvolt, Lilian Järvekülg, E. N. Dobrov
    Abstract:

    PotyViruses represent the most biologicAlly successful group of plAnt Viruses, but to our knowledge, this work is the first detAiled study of physicochemicAl chArActeristics of potyVirus virions. We meAsured the UV Absorption, fAr And neAr UV circulAr dichroism spectrA, intrinsic fluorescence spectrA, And differentiAl scAnning cAlorimetry (DSC) melting curves of intAct pArticles of A PotAto Virus A (PVA). PVA virions proved to hAve A peculiAr combinAtion of physicochemicAl properties. The intrAVirus coAt protein (CP) subunits were shown to contAin An unusuAlly high frAction of disordered structures, whereAs PVA virions hAd An Almost normAl thermAl stAbility. Upon heAting from 20°C to 55°C, the frAction of disordered structures in the intrAVirus CP further increAsed, while PVA virions remAined intAct At up to 55°C, After which their disruption (And DSC melting) stArted. We suggest thAt the structure of PVA virions below 55°C is stAbilized by interActions between the remAining structured segments of intrAVirus CP. It is not improbAble thAt the biologicAl efficiency of PVA relies on the disordered structure of intrAVirus CP.

  • in situ spAtiAl orgAnizAtion of PotAto Virus A coAt protein subunits As Assessed by tritium bombArdment
    Journal of Virology, 2001
    Co-Authors: Ludmila A. Baratova, E. N. Dobrov, Alexander V. Efimov, Alexander L. Ksenofontov, Natalija V Fedorova, Reet Hunt, G A Badun, L Torrance, Lilian Järvekülg
    Abstract:

    PotAto Virus A (PVA) pArticles were bombArded with thermAlly ActivAted tritium Atoms, And the intrAmoleculAr distribution of the lAbel in the Amino Acids of the coAt protein wAs determined to Assess their in situ steric Accessibility. This method reveAled thAt the N-terminAl 15 Amino Acids of the PVA coAt protein And A region comprising Amino Acids 27 to 50 Are the most Accessible At the pArticle surfAce to lAbeling with tritium Atoms. A model of the spAtiAl ArrAngement of the PVA coAt protein polypeptide chAin within the Virus pArticle wAs derived from the experimentAl dAtA obtAined by tritium bombArdment combined with predictions of secondAry-structure elements And the principles of pAcking α-helices And β-structures in proteins. The model predicts three regions of tertiAry structure: (i) the surfAce-exposed N-terminAl region, comprising An unstructured N terminus of 8 Amino Acids And two β-strAnds, (ii) A C-terminAl region including two α-helices, As well As three β-strAnds thAt form A two-lAyer structure cAlled An AbCd unit, And (iii) A centrAl region comprising A bundle of four α-helices in A fold similAr to thAt found in tobAcco mosAic Virus coAt protein. This is the first model of the three-dimensionAl structure of A potyVirus coAt protein.

Kimmo Rantalainen - One of the best experts on this subject based on the ideXlab platform.

  • structurAl flexibility Allows the functionAl diversity of potyVirus genome linked protein vpg
    Journal of Virology, 2011
    Co-Authors: Kimmo Rantalainen, Katri Eskelin, Peter Tompa, Kristiina Mäkinen
    Abstract:

    SeverAl virAl genome-linked proteins (VPgs) of plAnt Viruses Are intrinsicAlly disordered And undergo folding trAnsitions in the presence of pArtners. This property hAs been postulAted to be one of the fActors thAt enAble the functionAl diversity of the protein. We creAted A homology model of PotAto Virus A VPg And positioned the known functions And structurAl properties of potyvirAl VPgs on the novel structurAl model. The model suggests An elongAted structure with A hydrophobic core composed of AntipArAllel β-sheets surrounded by helices And A positively chArged contAct surfAce where most of the known Activities Are locAlized. The model most probAbly represents the fold induced immediAtely After binding of VPg to A negAtively chArged lipid surfAce or to SDS. When the chArge of the positive surfAce wAs lowered by lysine mutAtions, the efficiencies of in vitro NTP binding, uridylylAtion reAction, And unspecific RNA binding were reduced And in vivo the infectivity wAs debilitAted. The most likely uridylylAtion site, Tyr63, locAtes to the positively chArged surfAce. Surprisingly, A Tyr63AlA mutAtion did not prevent replicAtion completely but blocked spreAding of the Virus. BAsed on the locAlizAtion of Tyr119 in the model, it wAs hypothesized to serve As An AlternAtive uridylylAtion site. Evidence to support the role of Tyr119 in replicAtion wAs obtAined which gives A positive exAmple of the prediction power of the model. TAken together, our experimentAl dAtA support the feAtures presented in the model And the ideA thAt the functionAl diversity is AttributAble to structurAl flexibility.

  • interAction of A potyvirAl vpg with Anionic phospholipid vesicles
    Virology, 2009
    Co-Authors: Kimmo Rantalainen, Peter Astrup Christensen, Nisse Kalkkinen, Daniel E Otzen, Anders Hafrén, Kristiina Mäkinen
    Abstract:

    The virAl genome-linked protein (VPg) of PotAto Virus A (PVA) is A multifunctionAl protein thAt belongs to A clAss of intrinsicAlly disordered proteins. TypicAlly, this type of protein gAins A more stAble structure upon interActions or posttrAnslAtionAl modificAtions. In A membrAne lipid strip overlAy binding AssAy, PVA VPg wAs found to bind phosphAtidylserine (PS), but not phosphAtidylcholine (PC). According to circulAr dichroism spectroscopy, the secondAry structure of PVA VPg wAs stAbilized upon interActions with PS And phosphAtidylglycerol (PG), but not with PC vesicles. It is possible thAt this stAbilizAtion fAvored the formAtion of α-helicAl structures. Limited tryptic digestion showed thAt the interAction with Anionic vesicles protected certAin, otherwise Accessible, trypsin cleAvAge sites. An electron microscopy study reveAled thAt interAction with VPg substAntiAlly increAsed the vesicle diAmeter And cAused the formAtion of pore or plAque-like electron dense spots on the vesicle surfAce, which grAduAlly led to disruption of the vesicles.

  • PotAto Virus A genome linked protein vpg is An intrinsicAlly disordered molten globule like protein with A hydrophobic core
    Virology, 2008
    Co-Authors: Kimmo Rantalainen, Nisse Kalkkinen, Vladimir N Uversky, Perttu Permi, Keith A Dunker, Kristiina Mäkinen
    Abstract:

    Genome-linked protein VPg of PotAto Virus A (PVA; genus PotyVirus) hAs essentiAl functions in All criticAl steps of PVA infection, i.e. replicAtion, movement, And virulence. StructurAl feAtures of the recombinAnt PVA VPg were investigAted with the Aim to creAte An outline for structure-function relAtionships. CirculAr dichroism dAtA of PVA VPg reveAled A distinct neAr-UV spectrum indicAting thAt the environment Around its AromAtic residues is structured but rAther flexible, And A fAr-UV spectrum thAt wAs chArActerized by feAtures typicAl for intrinsicAlly disordered proteins. TemperAture-induced denAturAtion followed A typicAl All-or-none trAnsition whereAs ureA- And GdmHCl-induced denAturAtion proceeded viA A route best described by A three-stAte-model. The conclusion drAwn wAs thAt the overAll structure of PVA VPg is significAntly unstAble even in the Absence of denAturAnts. AcrylAmide fluorescence quenching And 1-Anilino-8-nAphthAlene sulfonAte binding experiments together with 1D And 2D NMR dAtA further verified thAt PVA VPg behAves As A pArtiAlly folded species thAt contAins A hydrophobic core domAin. Regions predicted to be disordered in PVA VPg were the ones thAt were cut the fAstest by trypsin whereAs regions predicted to be structured And to contAin the most conserved Amino Acids Among potyVirus VPgs were trypsin-resistAnt. Amino Acid composition AnAlysis of potyVirus VPgs reveAled A cleAr enrichment of disorder And depletion of structure-promoting residues. TAken together it seems thAt the nAtive structure of PVA VPg, And probAbly thAt of potyvirAl VPg in generAl, is A pArtiAlly disordered molten globule-like protein. Further experimentAtion is required to understAnd the functionAl regulAtion Achieved viA this property.