Restoration of potato virus X coat protein capacity for assembly with RNA after His-tag removal

Abstract

We found that the fusion of hexahistidine (H)6 tag to the potato virus X (PVX) coat protein (CP) abolished its ability to be assembled with viral RNA into helical nucleoprotein virus-like particles (VLPs). Instead, irregular agglomerates were produced upon incubation of PVX RNA with (H)6-tagged PVX CP. A factor Xa recognition site, IEGR, was inserted upstream of the CP coding sequence. Removal of the (H)6 tag from PVX CP by Xa protease restored its ability to bind RNA and to assemble VLPs. In addition to the canonical IEGR site, the factor Xa protease was found to cleave PVX CP at a second (non-consensus) site, AVTRGR, located close to the C-terminus of PVX CP. The latter cleavage did not affect reassembly of the PVX RNA and CP into VLPs.

A Recombinant Rotavirus Antigen Based on the Coat Protein of Alternanthera Mosaic Virus

“…For the mixtures where AltMV RNA was incubated with each of the proteins individually, the gel retardation assay revealed that the material was held up at the start of the gel. Data from our previous experiments suggest that the presence of histidine residues can lead to nonspecific aggregation of recombinant proteins with RNA [20]. This may explain the observation that RNA incubated with recombinant protein ER6 was retained in the gel at the start level.…”

The role of the 5′-cap structure in viral ribonucleoproteins assembly from potato virus X coat protein and RNAs

“…Instead, PVX RNA interaction with PVX TGBp1 turns the protein helix into a metastable state and further interaction with a ribosome triggers release of the free RNA, which results in RNA translation and formation of virus-specific products [15,20,31]. A defining role in this process is played by the conformational states of PVX CP and TGBp1 [32,33], so that translation of an RNA within a vRNP indicates that PVX CP of the vRNP has the native protein structure [15].…”

Proteínas de movimiento de la familia 30K:interacción con membranas biológicas y factores proteicos y su implicación en el transporte viral

“…De hecho, únicamente se ha descrito la interacción TGB1-CP para este grupo (Samuels et al, 2007). Hasta el momento existían dos modelos para explicar el transporte de Potexvirus a través de los PDs: uno en el que se postulaba que el PVX atravesaría el PD en forma de virión modificado (Santacruz et al, 1998) con la ayuda deTGB2 ,mediante su interacción con el genoma viral (Hsu et al, 2009) y otro en el que la partícula vírica consistiría en un complejo RNP formado por la TGB1, la CP y el vRNA (Lough et al, 2000), lo cual se asemejaría más a lo que ocurre con BMV o AMV (Karpova et al, 2006;Lukashina et al, 2009;Zayakina et al, 2009). Sin embargo, recientemente estudios basados en la utilización de virus quimera han esclarecido el papel que desempeña la CP en este proceso y han demostrado que la formación del virión es prescindible para el movimiento célula a célula pero necesaria para el movimiento sistémico (Betti et al, 2012).…”