Christiane Fritsch

Translation Products of Genome and Satellite RNAs of Tomato Black Ring Virus

Summary In wheat germ extracts and reticulocyte lysates the genome RNA molecules of tomato black ring virus (TBRV), RNA-1 (mol. wt. 2.8 × 106) and RNA-2 (mol. wt. 1.6 × 106), were translated into products of maximum mol. wt. 2.2 × 105 and 1.6 × 105, respectively. These products represent about 80% and 100% of the coding capacity of the two RNA species. The 1.6 × 105 mol. wt. polypeptide reacted with antiserum to TBRV particles but the translation products of RNA-1 did not; this is additional evidence that RNA-2 contains the coat protein cistron. Satellite RNA molecules (RNA-3, mol. wt. 4.8 × 105) associated with strain S of TBRV, like those associated with strain G, were translated in wheat germ extracts into a polypeptide of mol. wt. 4.8 × 104; this did not react with TBRV antiserum. Protease digestion released peptides from the translation product of strain S RNA-3 which were different from those released from the translation product of strain G RNA-3, suggesting that the two kinds of satellite RNA molecules differ in base sequence.

Translation of the Satellite RNA of Tomato Black Ring Virus in vitro and in Tobacco Protoplasts

Summary Translation of the satellite RNA (RNA-3) of tomato black ring virus (TBRV) in wheat germ extracts or reticulocyte lysates resulted in the synthesis of a polypeptide of mol. wt. about 48000, both in the presence and in the absence of RNA-1 and RNA-2. The RNA-3 specific polypeptide of TBRV strain G was slightly larger than that of strain S. A polypeptide of the same electrophoretic mobility as the in vitro translation product of RNA-3 was found in extracts of protoplasts infected with an isolate of TBRV-S possessing RNA-3 but not in extracts of protoplasts infected with an isolate lacking RNA-3. The lack of phenotypic effect of RNA-3 raises the question of the function of this protein in the infected plant.

Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

ABSTRACT RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.

Comparison of proteins synthesized in vivo and in vitro by mRNA from isolated protoplasts.

Studies of proteins synthesized in vitro by messenger RNA (mRNA) extracted from tobacco protoplasts showed that the changes in protein synthesis and especially the lack of certain proteins observed previously in isolated protoplasts did not result from a failure of translation.

Translation of tobacco rattle virus RNA in vitro using wheat germ extracts

Abstract When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.

Further studies on the translation products of tobacco rattle virus RNA in vitro.

Abstract Translation of RNA-1 (MW, 2.5 × 106) of tobacco rattle virus (TRV) strain PRN in wheat germ extracts containing spermidine resulted in the production of many polypeptides with a maximum MW of 170,000 and a clear band of 140,000. Translation of RNA-1 in lysates of rabbit reticulocytes resulted in these two products but little else. PRN-RNA-2 (MW, 1.0 x 106) was translated in both in vitro systems as two polypeptides, coat protein and a protein of MW 31,000 (band 1). Three bands of possible premature termination products were also produced in wheat germ extracts. The identity of coat protein was shown by coelectrophoresis in 10 and 3% polyacrylamide gels, specific aggregation with PRN protein, and coincidence between tryptic peptide fingerprints. Tryptic peptide mapping showed that band 1 was not a precursor for coat protein. There were, however, some peptides in digests of band 1 which coincided with coat protein peptides, which suggests a partial overlap of amino acid sequences. The relative abundance of coat protein and band 1 protein in translation products of PRN-RNA-2 in wheat germ extracts was dependent on the concentration of magnesium ion in the extracts. At concentrations suboptimal for total incorporation, translation resulted mainly in coat protein, whereas, at supraoptimal concentrations of magnesium, band 1 protein predominated in the translation products. We suggest that PRN-RNA-2 contains two open initiation sites.

Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

Comparison of the nucleotide sequences of five tomato black ring virus satellite RNAs

Summary The nucleotide sequences of the satellite RNAs associated with tomato black ring virus isolates L, G, E and C were determined and compared with the sequence of the satellite associated with strain S determined earlier. The sequence of satellite L closely resembled that of satellite S and both were to a lesser extent similar to satellites G, E and C which themselves were closely similar. There was 90% or more sequence homology between satellites in each group, G and E being the most closely related, with only one nucleotide (nt) difference between them. There was about 60% homology between the groups. Satellites G, E and C had longer 5′ non-coding regions (40 nt) than satellites L and S (14 nt), but were shorter by 11 to 13 nt in their 3′ non-coding regions. The sequences all contained a single large open reading frame for proteins of mol. wt. 47808 (424 amino acids) (L), 47792 (419 amino acids) (G, E) and 47698 (419 amino acids) (C). The deduced amino acid sequences differed principally in their N-terminal 65 residues. Several regions of sequence comprising 10 or more amino acids, and one of 31 amino acids, were identical in all satellite sequences. No marked sequence similarities were detected between the satellite sequences and a number of viral protein and nucleic acid sequences available in databases. The extent of homology between the 3′ ends of satellite sequences and those of RNA-1 or RNA-2 of TBRV was slight, consisting of several hexa- and heptanucleotides.

Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein.

Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.