The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

Abstract

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3= untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis. Tomato mosaic virus (ToMV) is a positive-strand RNA virus in the genus Tobamovirus, family Virgaviridae (1). The genome of a tobamovirus encodes at least four proteins (2). The 130K protein and its read-through product, the 180K protein, are involved in RNA replication and thus are referred to as replication proteins (3). The 130K protein contains the methyltransferase and helicase domains, and the read-through region of the 180K protein contains the polymerase domain. The other two tobamoviral proteins, movement protein and coat protein, are not required for RNA replication (4, 5).When the genomic RNA of a positive-strand RNA virus enters host cells, the replication proteins are translated from the genomic RNA, recognize the genomic RNA as a template for replication, and form replication complexes on intracellular membranes. Replication of all known eukaryotic positive-strand RNA viruses occurs in replication complexes formed on host membranes (6-10), which contain viral genomic RNAs, replication proteins, and host factors (11). To avoid elicitation of host defense systems triggered by viral double-stranded RNA (12), the activity of eukaryotic positive-strand RNA virus RNA-dependent RNA polymerases (RdRps) must be strictly regulated so that they synthesize negative-strand RNA only in the replication complexes that are sequestered from the cytoplasm. Many host factors, including chaperones and enzymes, and cellular membranes are hijacked by viruses for rep...

Replication of Tobamovirus RNA

“…At this point, the binding of replication proteins to the membrane is so strong that the association is maintained in a buffer containing 0.5-M (or even 1-M) NaCl (58,104), at which point most peripheral membrane proteins dissociate from the membranes. Through analysis of Tm-1, a protein inhibitor of ToMV RNA replication, it was suggested that the replication proteins first bind to membranes in a 0.5-M NaCl-sensitive manner and then form a complex with TOM1, TOM2A, and ARL8 to establish 0.5-M NaCl-resistant membrane binding (see Tm-1: An Inhibitor of Tobamovirus RNA Replication) (58). During these processes, the genomic RNA becomes resistant to nucleases in the absence, but not in the presence, of detergents (58).…”

Structural basis for the recognition–evasion arms race between Tomato mosaic virus and the resistance gene Tm-1

“…We proposed that ToMV-Hel must undergo a conformational change to interact with host factors during the assembly of the replication complex (27). In a cell-free replication system, Tm-1 inhibits the binding of replication proteins to the host membrane proteins TOM1 and ARL8 and the sequestration of replication templates, which are tightly linked to and precede the formation of the replication complex, in the membranous compartment (14). How partial inhibition of helicase NTPase activity affects overall replication, or if other mechanisms (e.g., inhibition of a conformational change required for replication complex assembly) also contribute to the inhibition by Tm-1 will be addressed in future studies.…”

“…In addition to the aforementioned domains, the 183-kDa protein also contains the RNA-dependent RNA polymerase domain in the read-through region. Tm-1 inhibits the formation of ToMV replication complexes on host intracellular membranes and consequently inhibits negative-strand viral RNA synthesis (14). The amino acid changes found in Tm-1 resistance-breaking mutants locate at the helicase domain of ToMV replication proteins (ToMV-Hel) and decrease the affinity of Tm-1 for the replication proteins (12).…”

Plant immunity against viruses: antiviral immune receptors in focus

“…The Tm-1-encoded product interacts directly with the viral replicase, impairing viral genome replication (Ishibashi and Ishikawa, 2013).…”