Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Structural flexibility allows the functional diversity of potyvirus genome-linked protein VPg.

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 ß-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.

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 alpha-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.

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, resembles a partially disordered molten globule. Further experimentation is required to understand the functional regulation achieved via this property.