Renata Grzela

Virulence Factor of Potato Virus Y, Genome-attached Terminal Protein VPg, Is a Highly Disordered Protein

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5′ end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.

A Novel Human Membrane Protein Interacting with the Short Fiber of Enteric Adenovirus

Human enteric adenoviruses of species F, HAdV-40 and HAdV-41 (Ad40 and Ad41), are associated with gastroenteritis in children. Ad attachment to the primary receptor on the cell surface is mediated by the distal head domain of the fiber protein, an antenna-like component of the adenovirus capsid. Differently from the majority of human Ads that possess one type of fiber on their capsid, the Ad40 and 41 have two distinct fibers. The long fiber recognizes the host membrane protein CAR, which permits virus attachment, but nothing is known about the role of the short fiber. Using the head domain of the Ad41 short fiber, we fished out a putative membrane protein that has never been previously described. This partner of the short fiber of enteric Ad41 (ParAd41) is a small, hydrophobic protein with three putative trans membrane domains, which interacts with the Ad41 short fiber but not with the Ad41 long fiber or with the fiber of respiratory Ad2 serotype. ParAd41 is localized in intracellular membranes including the nuclear membrane. Saturation of the short fiber with ParAd41 inhibits virus infectivity, which substantiates the putative role of ParAd41 in enteric Ads tropism. It is conceivable that the interaction of the short fiber with ParAd41 mediates virus postattachment endocytosis step as well as interaction with the nuclear membrane prior to the injection of viral DNA into the nucleus, thus enabling enteric adenovirus infection. This study is the first one to probe the molecular nature of enteric Ad41 tropism.

Peptides derived from plant virus VPg protein inhibit eIF4E oncogene

Viruses of the Potyviridae family have VPg protein covalently attached to the 5’ end of their linear RNA genome. The protein interacts with the host translation initiation factor eIF4E that occurs in plant cells in two isoforms, one being the preferable target of a given VPg, the remaining one still acting in host protein synthesis. In animal cells only one form of eIF4E is directly involved in protein synthesis. The human eIF4E is known to be an oncogene; elevated expression of eIF4E leads to oncogenic transformation, cancers in animal models and poor prognosis in human cancers, while reduction of the eIF4E level can reverse the transformed phenotype. We show that VPg protein delivery to cells containing only one eIF4E isoform involved in protein synthesis resulted in immobilization of eIF4E in the cytoplasm. The region of VPg involved in the interaction with eIF4E has been partially identified. Peptides derived from this region interacted better with eIF4E than complete VPg protein. Here we characterized one of VPg peptides, VPg5 and we show that VPg5 delivered to colon carcinoma HCT116 cells is able to inhibit cell growth, which is accompanied by reduction in eIF4E level. List of abbreviations AcMNPV Autographa californica multicapsid nucleopolyhedrovirus CBB Coomassie Brilliant Blue ClYVV Clover yellow vein virus eIF4E eukaryotic initiation translation factor 4E 4E BP, eIF4E binding protein FCS fetal calf serum LMV Lettuce Mosaic Virus MOI multiplicity of infection MW molecular weight ON overnight TMB 3,3,5,5-tetramethylbenzidine T. ni Trichoplusia ni cells PBST PBS buffer containing 0.05% Tween-20 pi post infection PI propidium iodide TEV tobacco etch virus TVMV tobacco vein mottling virus PVY potato virus Y VPg genome-linked viral protein