The citrus tristeza virus p33 protein functions as a viroporin.

Viroporins are viral proteins that form ion channels in the membranes of the host and, thus, alter the host ion homeostasis to create more favorable environments for the virus. Since the discovery of the ion channel activity of the M2 protein encoded by influenza virus A (species: Alphainfluenzavirus influenzae), many additional viral proteins have also been characterized as viroporins. However, most viroporins known thus far belong to animal viruses, while the discovery of plant virus viroporins has significantly lagged. In this work, we present evidence that the p33 protein, a membrane-associated protein of citrus tristeza virus (CTV; species: Closterovirus tristezae), possesses the characteristics of a viroporin. We first show the substantial structural similarities between the transmembrane and cytoplasmic domains of known Class I viroporins and those of the p33 protein. Using two-voltage electrode clamp assays in Xenopus oocytes, we further demonstrate the ion channel properties of p33 such as the ability to induce strong inward currents of potassium and sodium when activated at lowered membrane potentials. Finally, using confocal and electron microscopy, we show that, similarly to other Class I viroporins, the p33 protein triggers extensive membrane remodeling and discuss additional characteristics of p33 and the functions of this protein in the CTV infection, which resemble those found with viroporins of other viruses. This study represents the third report of a viroporin encoded by a plant virus and the first validation of the ability of a plant virus viroporin to induce currents across eukaryotic membranes using electrophysiological assays. The findings of this work open new avenues in research focusing on the understanding the role of viroporins in plant virus infections.