Movement Protein of a Closterovirus Is a Type III Integral Transmembrane Protein Localized to the EndoplasmicReticulum

Abstract

Cell-to-cell movement of beet yellows closterovirus requires four structural proteins and a 6-kDa protein (p6) that is a conventional, nonstructural movement protein. Here we demonstrate that either virus infection or p6 overexpression results in association of p6 with the rough endoplasmic reticulum. The p6 protein possesses a single-span, transmembrane, N-terminal domain and a hydrophilic, C-terminal domain that is localized on the cytoplasmic face of the endoplasmic reticulum. In the infected cells, p6 forms a disulfide bridge via a cysteine residue located near the protein's N terminus. Mutagenic analyses indicated that each of the p6 domains, as well as protein dimerization, is essential for p6 function in virus movement.Transport of plant viruses within and between cells is an active process that requires the function of virus-coded movement proteins (MPs). By definition, MPs are specialized proteins that are essential for the translocation of viral genomes or virions, but they are not required for virus genome replication or encapsidation. Viral MPs belong to several distinct protein families, each of which seems to exhibit a unique functional profile (10, 26). Many virus genera possess not one but two or three MPs. In addition, cell-to-cell movement of some viruses requires proteins whose primary functions are in genome replication or encapsidation (8,9,21).Among several present models of virus movement, two have approached canonic status (10, 26). One is a Tobacco mosaic virus (TMV) model (4). The only TMV MP, the 30-kDa protein p30, is able to bind viral RNA and guide it through the plasmodesmata (13). Its additional activities include modification of plasmodesmatal gating properties and interactions with microtubules, actin microfilaments, endoplasmic reticulum (ER) (28,31,38), and a cell wall-specific host enzyme (12). However, the exact mechanistic contributions of these MP associations to intracellular movement are a matter of debate (6,17,41). Likewise, the transport mechanism of the RNA-MP complex through plasmodesmata largely remains a mystery. The leading model proposes that TMV-type MPs recruit a preexisting host machinery for intercellular trafficking (19,27). Interestingly, both rod-shaped RNA viruses related to TMV and several icosahedral RNA and single-stranded DNA viruses appear to follow this movement paradigm (16, 26).The second well-recognized model applies to several families of the icosahedral RNA viruses and pararetroviruses (35,42). The MPs of these viruses reorganize plasmodesmata by inducing formation of the tubules through which mature virions translocate from cell to cell. The MP and endomembrane secretion system appear to be sufficient for tubule formation, whereas intact cytoskeleton is required for proper positioning of the tubules relative to plasmodesmata (24).Mounting evidence indicates that the filamentous potexviruses do not fit in any of the abovementioned models. The 25-kDa MP (p25) of Potato virus X (PVX) possesses nucleoside triphosphatase and RNA helicase activitie...

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

“…Among plant viruses, cell-to-cell transport depends on one or more virus-encoded MPs, and many of these interact with membranes. In those viruses encoding more than one small MP, there is experimental evidence demonstrating that at least one of the encoded MPs is an integral membrane protein (22,29,41). For the virus encoding only one large MP (30K superfamily), a topological model for TMV MP with two HRs spanning the membrane has been proposed (4).…”

“…Cultures of Arabidopsis tumefaciens strain C58C1 transformed with the different binary pMOG800 plasmids were used to infiltrate Nicotiana benthamiana plants as described previously (12). Three days postinfiltration, the leaves were processed to obtain enriched membranous fractions as described previously (29). The resultant membranous enriched pellet was resuspended in buffer A (20 mM HEPES, pH 6.8, 150 mM potassium acetate, 250 mM mannitol, 1 mM MgCl 2 , 2.5 ml of protease inhibitor cocktail for plant cell and tissue extracts; Sigma) and divided into three aliquots for the untreated, alkaline wash, or urea treatments as described previously.…”

“…3A, untreated lanes) upon centrifugation of the microsome-containing translation reaction mixture, indicating that it could be either a membraneassociated (peripheral or integral) protein or a lumenally translocated protein. To differentiate between these possibilities, the translation reaction mixtures were first washed with sodium carbonate (pH 11.5), which renders microsomes into membranous sheets, releasing the soluble luminal proteins (25,29). As shown in Fig.…”

“…There are two TM segments in the p9 protein of carnation mottle virus (41), whereas the p6 protein of beet yellow virus (29) and the p7B protein of melon necrotic spot virus (22) have a single TM segment. In viruses with genomes that include three partially overlapping open reading frames, termed the triple-gene block (TGB), all three TGB proteins are required for movement where the two smaller proteins, TGBp2 and TGBp3, are also TM proteins (24).…”

The Complex Genetics of Citrus tristeza virus

“…Viral mutants with a deletion in the p6 and p20 ORFs failed to infect citrus plants systemically, suggesting their possible roles in virus translocation or infection of the whole plant. Likewise, the p6 homologue in BYV is a movement protein [65], and similarly to homologous proteins function in BYV [11], CP, CPm, HSP70h and p61 probably participate in the viral movement. CTV genome has several ORFs that are non-conserved in the family Closteroviridae, thus unique for CTV.…”