The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3

Abstract

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183-and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a templatedependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.

Host Factors in Positive-Strand RNA Virus Genome Replication

“…A 41-kDa subunit of eIF3 copurified with BMV RdRp and in-teracted with BMV 2a polymerase, and addition of purified eIF3 or the 41-kDa subunit stimulated negative-strand synthesis by BMV RdRp (28). TMV RNA replication proteins interacted with and coimmunoprecipitated with the host GCD10 subunit of eIF3, and anti-GCD10 antibodies inhibited negative-strand synthesis by TMV RdRp (26,36). Such host factors may have functions related to the aminoacylatable 3Ј tRNAlike elements of TMV and BMV genomic RNAs (where negative-strand RNA synthesis initiates), to the tRNA T⌿C stemloops of the BMV RE template-recruitment signals, or to both.…”

Viral and Cellular Proteins Involved in Coronavirus Replication

“…2. Translation factors: elongation factors EF-1a (Blackwell and Brinton 1997;Harris et al 1994;Joshi et al 1986), -b and -g (Das et al 1998), EF-Tu (Blumenthal and Carmichael 1979), and eukaryotic initiation factor eIF-3 (Osman and Buck 1997;Quadt et al 1993). 3.…”

Rna Viruses Redirect Host Factors to Better Amplify Their Genome

“…Hence, the host components of the two viral polymerases correspond to different subunits of eIF3. Antibodies against the 56 kDa subunit inhibit TMV RNA synthesis in vitro (Osman and Buck, 1997).…”