A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

Abstract

Systematic deletion and peptide tagging of the amino-terminal domain (NT, ϳ43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5 end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.Zucchini yellow mosaic potyvirus (ZYMV) is a member of the Potyviridae family, the largest group of plant-infecting viruses (31). As in all potyviruses, the ZYMV genome consists of a single messenger-polarity RNA molecule of about 10 kb, encapsidated by ϳ2,000 subunits of coat protein (CP), forming a helical, flexuous filament particle about 750 nm long and 11 nm wide (8,19).Though there are no high-resolution X-ray diffraction data available on the structure of potyvirus CP, there is a considerable amount of information about its topology. Structure predictions, together with immunological studies (7, 29) of potyvirus CPs, have demonstrated structural features similar to those of the CP of tobacco mosaic virus (21) and potato virus X (26). Like those proteins, potyviral CP is a three-domain protein with variable N-and C-terminal regions exposed on the virion surface and a conserved core domain that probably interacts with viral RNA (1, 29). ZYMV CP (279 amino acids [aa]) is composed of a 214-aa core domain flanked by 43-to 45-and 20-aa N-and C-terminal domains, respectively, as predicted by Shukla et al. (30). The putative trypsin protease motif of potyvirus CP, representing the end of the surface-exposed N-terminal domain (NT), is presumed to be positioned between amino acids Lys and Asp, located in the KDKD motif ...

Characterization of a Mitogen-Activated Protein Kinase Gene from Cucumber Required for Trichoderma-Conferred Plant Resistance

“…Therefore, we modified the expression levels of TIPK by employing an attenuated potyvirus vector, ZYMV-AGII (Arazi et al, 2001). This vector represents a unique system for gene expression in cucurbit cytosol.…”

“…Because it was engineered from an attenuated strain, it is nonpathogenic and does not impair growth of the host plant (Arazi et al, 2001). The AGII-GFP construct has been reported to be stable in cucumber plants for at least 60 dpi (Arazi et al, 2001), and we also validated the propagation of the virus during our experimental period. We determined the stability of the viral constructs throughout the experiments (Fig.…”

“…ZYMV-AGII is a potyvirusbased vector system that has been successfully used for overexpression of various foreign genes in cucurbits (Arazi et al, 2001;Aly et al, 2005). In contrast to other known viral vectors, which cause severe disease to host plants, the AGII vector was created from an attenuated engineered ZYMV potyvirus (Arazi et al, 2001) and does not elicit the severe phenotype or developmental impairment caused by wild-type virus, and no symptoms are developed in cucumbers (Gal-On and Raccah, 2000). Therefore, it is attractive for overor down-regulation of endogenous gene expression.…”

Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

“…To our knowledge, neither the insertion site used nor specific affinity tag-based purification of potyviral VPg has been described previously. Epitope-tag insertions coupled with purification have been reported for the potyviral proteins HC-Pro, 6K2 and CP (Restrepo-Hartwig & Carrington, 1994;Blanc et al, 1999;Arazi et al, 2001). Successful purification under native conditions showed that the affinity tag, and hence the C terminus of VPg, was exposed on the surface of both VPg and NIa.…”

Desarrollo De Un Vector Para La Expresión Simultánea De Múltiples Proteínas en Plantas Basado en El Potyvirus Del Grabado Del Tabaco

“…Además de dichos estudios básicos, estos virus también se han utilizado para expresar en plantas genes foráneos de diferentes tipos. Por ejemplo, con el virus del mosaico amarillo del calabacín (Zucchini yellow mosaic virus, ZYMV) se ensayó la expresión de un epítopo inmunogénico del virus de la fiebre aftosa insertado en el área amino terminal de la proteína de la cápside (evaluando variadas deleciones dentro de la misma), y se ha comprobado que dicha región no es necesaria para el movimiento sistémico viral (Arazi et al, 2001a). Asimismo, con una cepa atenuada de este virus se ha probado la expresión del gen del interferón alfa-2 humano en plantas de calabaza y pepino (Arazi et al, 2001b), y también la adquisición de resistencia a herbicidas que contienen glufosinato de amonio en plantas de melón, pepino, calabaza y sandía gracias a la expresión del gen bar, que codifica para la fosfinotricina acetiltransferasa (Shibolet et al, 2001).…”