Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

Abstract

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNAdependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positivestrand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130-and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the nonmembrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.The virus particles of positive-strand RNA [(ϩ)RNA] viruses contain single-stranded, messenger-sense genomic RNA, and these viruses replicate via complementary, negative-strand RNA [(Ϫ)RNA] templates. Most plant viruses and many animal viruses are (ϩ)RNA viruses. After infection, the genomes of these viruses are translated to produce viral proteins that function in viral RNA replication. The replication of eukaryotic (ϩ)RNA viruses takes place in replication complexes that are formed on the intracellular membranes of infected cells and that contain the viral RNA replication proteins and endogenous (Ϫ)RNA templates. The replication complexes synthesize viral (ϩ)RNAs from ribonucleoside triphosphates (rNTPs) and release them into the cytoplasm (4, 24).The genus Tobamovirus, which belongs to the alphavirus-like superfamily of (ϩ)RNA viruses, includes Tobacco mosaic virus and Tomato mosaic virus (ToMV) and many related viruses. The genome of a tobamovirus is a 6.4-kilobase RNA that encodes a nonstructural protein with an approximate molecular mass of 130 kDa (130,000-molecular-weight [130K] protein), its read-through product of 180 kDa (180K prote...

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

“…This feature may be analogous to the case of the 130-kD replication protein (130K) of tomato mosaic tobamovirus (ToMV), which also functions as a VSR (Hagiwara-Komoda et al, 2008). While the membrane-associated form of 130K is involved in viral replication, the residual soluble form of 130K suppresses RNA silencing in the cytoplasm (Nishikiori et al, 2006). Since ToMV 130K has been suggested to possess optimal affinity with membrane to maintain balanced accumulation of its membranebound form and soluble form, TGBp1 may be properly regulated to promote efficient viral propagation.…”

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis

“…After tRNA transfer, eEF1A is recycled to its active GTP-bound form by the guanine nucleotide exchange factor, eEF1B. eEF1A interacts with the RNA dependent RNA polymerases (RdRp) of tobacco mosaic virus and turnip mosaic virus and is known to be associated with the replication complexes of potyviruses, tobamoviruses, and tombusviruses (Yamaji et al, 2006;Nishikiori et al, 2006;Thivierge et al, 2008;Li et al, 2010b). We also show that SMV replication is associated with the unfolded protein response (UPR) and that eEF1A contributes to this.…”

An inhibitor of viral RNA replication is encoded by a plant resistance gene

“…ToMV RNA replication occurs in membrane-bound complexes (16)(17)(18). When ToMV RNA is translated in membrane-depleted BYL (mdBYL), the 130K and 180K replication proteins are produced, but ToMV-related RNA is not synthesized.…”