Abstract
Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses.
Highlights
Positive-strand RNA [(+)RNA] viruses—the largest class of viruses, include significant pathogens of humans, animals, and plants (King et al, 2012)
Polyclonal antisera raised against the Turnip yellow mosaic virus (TYMV) 66-kDa protein (66K) protein, the proline-rich region (PRR) domain shared by 140 kDa (140K) and 98K proteins and the TYMV capsid were described previously (Prod’homme et al, 2001; Jakubiec et al, 2004), and were used at dilutions of 1/2,000, 1/8,000, and 1/50,000, respectively
To test the membrane association properties of TYMV replication proteins during viral infection, TYMV-infected Chinese Cabbage tissues were fractionated by centrifugation to recover a membrane pellet fraction containing viral replication complex (VRC) (Prod’homme et al, 2001; Jakubiec et al, 2004), which was analyzed by Western blot using specific antibodies raised against the 66K protein (Prod’homme et al, 2001) and the PRR domain shared by 140K and 98K proteins (Jakubiec et al, 2004) (Figure 1)
Summary
Positive-strand RNA [(+)RNA] viruses—the largest class of viruses, include significant pathogens of humans, animals, and plants (King et al, 2012) Replication of their genome requires the assembly of an intricate viral replication complex (VRC) comprising both viral and host proteins (reviewed in Nagy and Pogany, 2011; Wang, 2015). A universal feature of (+)RNA VRCs is their close association with intracellular membranes (Buck, 1996; Salonen et al, 2005; Grangeon et al, 2012), resulting in massive viralinduced membrane rearrangements and/or proliferation These host-derived membranes, which anchor the components of the replication complex, are thought to create a favorable environment for RNA synthesis by concentrating crucial viral and host factors, and possibly protecting the viral RNA progeny from host cell antiviral surveillance system. Despite great advances in imaging of these subcellular structures, characterisation of their ultrastructural details, and identification of some of the host factors or cellular pathways involved in their formation, all of which has revealed many similarities among (+)RNA VRCs (reviewed in den Boon and Ahlquist, 2010; Belov and van Kuppeveld, 2012; de Castro et al, 2013; Harak and Lohmann, 2015), we are still far from understanding the molecular details of this membrane association, and how viral replication factors target, bind, and remodel membranes of specific cell organelles during VRC biogenesis
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