Immunodetection, Expression Strategy and Complementation of Turnip Crinkle Virus p28 and p88 Replication Components

Abstract

The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5' end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs the synthesis of polypeptides of approximately 28 and 88 kDa, (ii) the 88-kDa protein is immunologically related to p28, consistent with p88 being a readthrough product, and (iii) p28, but not p88, is detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a beta-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5'-proximal open reading frame for any member of the family Tombusviridae.

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…These results are in agreement with those reported for other members of the family Tombusviridae which show that, besides the RdRp, the corresponding virus produces an additional, relatively small polypeptide that is indispensable for effective pathogen multiplication. The lack of complementation among p27stop and p27tyr mutants, which are impaired in p86 and p27 synthesis, respectively, suggests either a cis-preferential requirement for both viral replicase proteins and/or a coupling between translation and replication, as proposed for several members of the Tombusviridae [Okamoto et al, 2008;Oster et al, 1998;White et al, 1995] and also other viral families [e.g. Lewandowski & Dawson, 2000;Neeleman & Bol, 1999].…”

“…It should nevertheless be noted that, within the family Tombusviridae, only the RdRp functioned in a cis-preferential manner in the case of Red clover necrotic mosaic virus (genus Dianthovirus) [Okamoto et al, 2008], whereas the opposite situation (i.e. cis-preferential functioning of ORF1 product) was reported for TCV and Tomato bushy stunt virus (TBSV; genus Tombusvirus) [Oster et al, 1998;White et al, 1995]. Besides the probability that PFBV p27 and p86 do not function well in trans, the failure of p27-and p86-deficient PFBV mutants to accumulate in complementation assays might also be because (i) the tyrosine substitution introduced in p27tyr is not the best replacement for p86, (ii) the stoichiometry of p27 and p86 is suboptimal and/or (iii) the nucleotide changes introduced in each case lead to disruption of cis-acting elements.…”

“…Este aspecto se ha confirmado también en otros componentes de la familia, como el virus del mosaico necrótico del trébol rojo (Red clover necrotic ringspot virus, RCNMV) (género Dianthovirus) [Bates et al, 1995;Takeda et al, 2005;Okamoto et al, 2008], el virus del mosaico del Panicum (Panicum mosaic virus, PMV) (género Panicovirus) [Batten et al, 2006], la cepa D del virus de la necrosis del tabaco (Tobacco necrosis virus D, TNV-D) (género Necrovirus) [Molnár et al, 1997], en dos carmovirus, el virus del arrugamiento del nabo (Turnip crinkle virus, TCV) y el virus de las manchas necróticas del melón (Melon necrotic spot virus, MNSV) [Hacker et al, 1992;White et al, 1995;Genovés et al, 2006], y en el virus del arabesco del Pelargonium (Pelargonium line pattern virus, PLPV), propuesto como miembro de un nuevo género, Pelarspovirus, dentro de la familia Tombusviridae [Castaño et al, 2009].…”

“…Así, normalmente, esta última se acumula de 10 a 20 veces menos en tejido infectado, que el producto de la ORF 1 [Hacker et al, 1992;Lupo et al, 1994;White et al, 1995;Scholthof et al, 1995b;Panaviene et al, 2004]. La actividad enzimática de polimerización asociada a la RdRp ha sido estudiada, tanto in vitro como in vivo, para la RdRp de distintos miembros de la familia incluyendo varios tombusvirus [Nagy & Pogany, 2000;Panavas et al, 2002a;Panaviene et al, 2004], un dianthovirus [Bates et al, 1995] y un carmovirus [Rajendran et al, 2002].…”

“…La ORF 1 codifica un producto de 26-28 kDa, cuya lectura a través permite la traducción de la ORF 2, dando lugar a una RdRp de entre 86 y 89 kDa [Hacker et al, 1992;White et al, 1995;Wang & Wong, 2004]. Todos los carmovirus secuenciados poseen dos pequeñas ORF centrales (ORF 3 y 4), normalmente solapadas, que codifican una pareja de proteínas que, al menos en algunas especies virales, se ha confirmado que se complementan en trans durante el movimiento viral y cuyos tamaños oscilan entre 7 y 8 kDa, en el caso del péptido más pequeño, y de 8 a 12 kDa, en el de mayor tamaño.…”