Potyviruses represent one of the most economically important and widely distributed groups of plant viruses. Despite considerable progress towards understanding the cellular and molecular basis of their pathogenicity, many questions remain about the mechanisms by which potyviruses suppress host defences and create an optimal intracellular environment for viral translation, replication, assembly and spread. The review focuses on the multifunctional roles of potyviral proteins and their interplay with various host factors in different compartments of the infected cell. We place special emphasis on the recently discovered and currently putative mechanisms by which potyviruses subvert the normal functions of different cellular organelles in order to establish an efficient and productive infection.

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.

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 ofPotato virus AVPg 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 β-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 ofin vitroNTP binding, uridylylation reaction, and unspecific RNA binding were reduced andin vivothe 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.

We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.

Asymmetrical flow field-flow fractionation (AF4) separates sample components based on their sizes in the absence of a stationary phase. It is well suited for high molecular weight samples such as virus-sized particles. The AF4 experiment can potentially separate molecules within a broad size range (~10³−10⁹ Da; particle diameter from 2 nm to 0.5−1 μm). When coupled to light scattering detectors, it enables rapid assays on the size, size distribution, degradation, and aggregation of the studied particle populations. Thus, it can be used to study the quality of purified viruses and virus-like particles. In addition to being an advanced analytical characterization technique, AF4 can be used in a semi-preparative mode. Here, we summarize and provide examples on the steps that need optimization for obtaining good separation with the focus on virus-sized particles.