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Title: Identification and functional characterization of P1N-PISPO, a new gene product present in sweet potato potyviruses
Author: Mingot Martí, Ares
Director/Tutor: López Moya, Juan José
Ferrer i Prats, Albert
Keywords: Virologia molecular
Molecular virology
Sweet potatoes
Issue Date: 31-May-2016
Publisher: Universitat de Barcelona
Abstract: [eng] Sweet potato feathery mottle virus (SPFMV) (Potyvirus genus, Potyviridae family) causes important yield losses in sweet potato crops, in particular in co-infections with the unrelated crinivirus Sweet potato chlorotic stunt virus (SPCSV). This thesis addresses the characterization of some novel aspects in the infectious cycle of SPFMV, such as the expression, production and function of a new gene product named P1N-PISPO. A better understanding of SPFMV genome organization and the functions of their gene products might be relevant to improve the control strategies against this virus and the associated diseases in sweet potato crops. The positive-sense RNA genome of SPFMV contains a large ORF, translatable as a polyprotein yielding a set of functional mature gene products (P1, HCPro, P3, 6K1, CI, 6K2, VPg-Nla, Nlb and CP), and a short ORF named PIPO in the -1 frame, embedded within the P3 region. In addition to this organization, common to all the members of the Potyvirus genus, another ORF named PISPO was predicted in the genome. PISPO is in the -1 frame within the P1 region of SPFMV and other related potyviruses, starting at a conserved G1_2A6_7 motif, similar to the motif found upstream of PIPO. The expression of PISPO during SPFMV viral infection could result in the production of a putative new gene product P1N-PISPO. In the present work, the presence of the PISPO frame has been investigated in a Spanish isolate of SPFMV infecting Ipomoea batata plants. The genome sequence of this isolate has been assembled from NGS data, showing that the expected trans-framed PISPO sequences is present, preceded by a G2A6 motif. A specific analysis of the NGS data has revealed a significant proportion of transcripts with an extra A in the motif at the beginning of PISPO, as well as a lower proportion of transcripts with an extra in the corresponding conserved motif preceding the PIPO region. These results have demonstrated that a polymerase slippage mechanism could generate transcripts containing extra A residues (G2A7) to allow the translation of P1N-PISPO and P3N-PIPO gene products. Analysis of the viral gene products present in SPFMV infected plant tissues has been performed using LC-MS/MS after separation in SDS- PAGE, focusing in products > 50KDa. Peptides corresponding to the P1 protein have been detected from both the N-terminal portion (11 different peptides, 39% coverage), before the frameshifting signal and therefore common for P1 and P1N-PISPO, and in the C- terminal part (2 peptides exclusive for P1, 10% coverage). Interestingly, four peptides exclusive of PISPO, in its unique ORF (21.3% coverage), have been also found. These results have confirmed that both products P1 and P1N-PISPO are expressed and coexisted during SPFMV infection. Furthermore, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana have revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, the results of this thesis have confirmed the expression of P1N-PISPO during SPFMV infection and they have revealed a polymerase slippage mechanism as the responsible of P1N-PISPO production. Our results also have demonstrated the role of P1N-PISPO as a RNA silencing suppressor.
[cat] El virus Sweet potato feathery mottle virus (SPFMV) (Gènere Potyvirus, Família Potyviridae) presenta un genoma de cadena senzilla i positiva de ARN que conté una pauta de lectura oberta (ORF), traduïda com una poliproteïna que dóna lloc a un conjunt de productes gènics madurs, i una ORF curta anomenada PIPO que es troba en el marc de lectura -1 de la regió P3 de la poliproteïna. A més a més, en el seu genoma s'ha observat una ORF addicional anomenada PISPO. PISPO es troba en el marc de lectura -1 dins de la regió gènica de la P1 en SPFMV i els potyvirus més propers, començant en el motiu conservat G 1_2A6_7 i similar al motiu d'inici de PIPO. La expressió de PISPO durant la infecció del virus donaria lloc a la producció del hipotètic producte gènic P1N-PISPO. La presència de la pauta de lectura PISPO, precedida per un motiu G2A6, ha estat confirmada en un aïllat Espanyol de SPFMV. L'anàlisi de dades obtingudes per seqüenciació massiva, ha permès identificar una proporció significativa de transcrits que contenen una A extra en el motiu al començament de PISPO, així com una proporció menor de transcrits amb aquesta A extra en el motiu de PIPO. Aquests resultats han demostrat que un mecanisme d'edició de la polimerasa podria generar transcrits amb residus A extra (G2A7), els quals permetrien la traducció dels productes gènics P1N-PISPO i P3N-PIPO. L'anàlisi del productes gènics virals presents en teixits de plantes infectades amb SPFMV s'ha dut a terme mitjançant un experiment de LC-MS/MS. Pèptids corresponents a la part N-terminal de la proteïna P1 (abans del motiu d'edició) han estat detectats, així com pèptids exclusius de la part C-terminal de la P1 i de la pauta de lectura de PISPO. Aquests resultats han confirmat que tant P1 com P1N-PISPO són expressats i co-existeixen durant la infecció de SPFMV. L'expressió transitòria de productes gènics de SPFMV coagroinfiltrats amb un gen reporter en Nicotiana benthamiana ha revelat que P1N-PISPO actua com a supressor de silenciament d'ARN, un rol associat normalment a HCPro en altres potyvirus. Per últim, la mutació de motius WG/GW presents en P1N-PISPO aboleix la seva activitat com a supressor, suggerint que la funció pot està lligada a la interacció amb les proteïnes Argonauta de la maquinària de silenciament, tal i com passa en altres supressors virals.
Appears in Collections:Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Farmàcia)

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