In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus)

Abstract

The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model.

FIG. 1

(A) Genome organization of figwort mosaic virus (FMV). The circular double-stranded DNA of 7743 base pairs is indicated by the interwoven lines. The inner circle shows the position of selected restriction sites. The two RNA transcripts are indicated by lighter inner lines. The single interruption in the minus strand (designated α) and three in the complementary strand (designated β ₁, β ₂, and β ₃) are indicated. The peripheral arrows and Roman numerals indicate the location of the open reading frames. The two intergenic regions, the smaller one between genes V and VI [shown as S-IR in (B)] and the larger one between genes VI and VII [shown as L-IR in (B)], contain promoters for gene VI mRNA and the larger full-length transcript, respectively. (B) Positions of the chloramphenicol acetyltransferase (CAT) gene insertions into the cloned genome of figwort mosaic virus. These DNAs were inserted into a transcription vector (pGEM-3Z or pGEM-4Z) and used for making artificial transcripts of the viral genome. The T7 promoter and the viral genome are shown as the hatched retangular bar. The position of unique restriction sites (Sac I, Stu I, and Sph I) used to linearize the plasmid constructs for the preparation of runoff transcripts driven from the T7 promoter are indicated. Constructs with asterisks indicate that the CAT gene is positioned as a separate cistron with its own start codon.

FIG. 2

Proteins synthesized in lysates in response to RNA transcripts containing gene VI of figwort mosaic virus. Reticulocyte lysate reaction mixtures, after treatment with microccal nuclease, were incubated for 1 h at 30°C in the absence of added mRNA (lanes 1 and 5, or gene VI transcripts generated from pGEM VI (lanes 2 and 6) or full-length transcripts generated from pH54 (lane 7). Some aliquots were removed and analyzed directly by Western blotting (lanes 1–4) or proteins were immunoselected with antibody to FMV gene VI antibody and Pansorbin, followed by autoradiography (lanes 5 and 6). In the Western blots the filters were treated with gene VI antibody and followed by goat anti-rabbit IgG linked to alkaline phosphatase (14). Lane 3 consists of protein from FMV inclusion bodies isolated from infected plants. Lane 4 had similar extracts from healthy plants. The positions of stained proteins of various molecular weights are indicated on the right margin.

FIG. 3

Assay of CAT enzyme in lysate protein synthesis reaction mixtures incubated with transcripts prepared from various plasmid constructs. Protein synthesis reaction mixture (30 μl) was incubated at 30°C for 1 h with transcripts prepared from various plasmid constructs. Aliquots (5 μl) were diluted with 45 μl of CAT enzyme assay buffer Tris-HCl (pH 7.9), 0.25 M, and then assayed for CAT enzyme as described previously (10). The thin-layer plate was autoradiographed. Lane 1, control without any transcript; lane 2, CAT enzyme standard synthesized in vitro in lysate from a pGEM-CAT construct; lane 3, pH75 transcript; lane 4, pH53(8) transcript; lane 5, pH55 transcript; lane 6, pH54 transcript; lane 7, transcript from pH54 linearized with StuI; and lane 8, pH52 transcript. Data in lanes 6–8 are from a different experiment. Hence, the lanes do not match exactly with lanes 1–5. For convenience, the lane numbers from the autoradiogram are provided under each plasmid construct.

FIG. 4

Northern blot analysis of pH52 transcript incubated in lysate protein synthesis reaction mixture was performed as described in the Materials and Methods section. Lane 1, no transcript added; lane 2, transcript from pH52 but maintained at 0°C; and lane 3, transcript from pH52 incubated at 37°C for 30 min. Positions of 16S and 23S rRNA from E. coli are indicated by arrows.

FIG. 5

Hybrid arrest during cell-free synthesis of CAT in lysates in response to FMV transcripts from viral genomes containing CAT gene insertions. Aliquots of lysate reaction mixtures with various transcripts were assayed for CAT enzyme activity. Lane 1, no transcript added; lane 2, CAT enzyme standard; lane 3, transcripts from pH54; lane 4, transcripts from pH54 subjected to hybrid arrest with antisense RNA of the 5′ leader of the full-length transcript; lane 5, transcripts from pH52; lane 6, transcripts to pH52 subjected to hybrid arrest with antisense RNA to the 5′ leader of the full-length transcript. For convenience, lane numbers from autoradiogram are provided under each plasmid construct.