Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

Abstract

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pittingassociated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic a helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

“…Most positive single-strand RNA viruses form VRCs in association with ER, but which cell membranes are utilized by Betaflexiviridae for replication, have not been clarified yet. Recently, the association of the replicase protein with the host-cell ER was reported for GRSP-aV (Prosser et al 2015 ).…”

Genetic diversity and identification of putative recombination events in grapevine rupestris stem pitting-associated virus

“…The genome structure is identical for all isolates for which the full genome sequences are available (Giampetruzzi et al, 2012;Hu et al, 2015;Lima et al, 2009Lima et al, , 2006aLima et al, , 2006bMeng et al, 2005Meng et al, , 1998Morelli et al, 2011;Poojari et al, 2013;Zhang et al, 1998). Using autofluorescent protein tagging and fluorescence microscopy, the subcellular localisation of protein products of all five ORFs have been described (Meng and Li, 2010;Prosser et al, 2015;Rebelo et al, 2008). The majority of the genome (nt position 61-6546; GenBank accession AF057136) consists of ORF 1, encoding a 244 kDa polyprotein containing elements involved in virus genome replication and mRNA transcription.…”

“…OTU and AlkB are almost exclusively observed in RNA viruses that infect woody perennials, leading to the assumption that the two domains play an active role in the process with which these viruses infect woody plants (Bratlie and Drabløs, 2005;Makarova et al, 2000;Meng and Rowhani, 2017). Presumably, one or both of the cysteine protease domains are also involved in the cleavage of the replicase polyprotein, assisting in the formation of functional replication machinery (Makarova et al, 2000;Meng et al, 1998;Zhang et al, 1998 (Buck, 1996;Nagy and Pogany, 2008;Prosser et al, 2015;Waigmann et al, 2007). The triple gene block (TGB1, TGB2 and TGB3) comprises ORFs 2, 3 and 4, and is observed in members of the genera Foveavirus and Carlavirus.…”

Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation

“…We next tested whether the membrane-associated EMR2-NTF can be extracted from cell membrane by 1 M NaCl (high salt) or 100 mM Na 2 CO 3 , pH 11 (high pH), conditions usually used to dissociate some peripheral membrane proteins 46 . As shown, the EMR2-NTF was not solubilized by high salt or high pH treatment of cells expressing EMR2-WT-myc (Fig.…”

“…The supernatant was collected and centrifuged for 60 min at 100,000 g (35,000 rpm; rotor SW50.1; Beckmann) to give a crude membrane pellet and cytosol supernatant. For further extraction of peripheral proteins 46 , the crude membrane pellets were treated with 1 M NaCl or 100 mM Na 2 CO 3 , pH 11 overnight and then ultra-centrifuged at 100,000 g for 60 min at 4 °C.…”