Secondary Structures in the Capsid Protein Coding Sequence and 3′ Nontranslated Region Involved in Amplification of the Tobacco Etch Virus Genome

The plant potyviruses are members of the picornavirus supergroup of positive-strand RNA viruses. A typical potyvirus, such as tobacco etch virus (TEV), contains a single-component RNA genome of approximately 10 kilobases that encodes a large polyprotein whose processing is catalyzed by three virus-encoded proteinases (21) (Fig. ​(Fig.1A).1A). The single capsid protein (CP) (263 amino acid residues) is encoded by sequences at the 3′ end of the open reading frame and, with genomic RNA, forms a flexuous rod-shaped virion of 700 to 800 nm in length (24). Based on mutational and biochemical analyses, all of the potyvirus-encoded proteins, except CP, were shown to be necessary for efficient genome replication (5, 11, 13, 15–17, 20, 23, 26). FIG. 1 Genetic organization of the TEV genome and CP-coding region. (A) Diagrammatic representation of the TEV genome. Proteins encoded by the designated regions are indicated above the map. Vertical lines indicate sequence encoding polyprotein processing sites. ... Despite the dispensability of the CP for TEV genome replication, two cis-active properties of the CP-coding region have been identified. First, ribosomes must be able to traverse the CP-coding sequence to a point between codons 138 and 189 (TEV nucleotides 8932 to 9084) (Fig. ​(Fig.1B).1B). Inhibition of translation through the 5′ region of the CP sequence by introduction of stop codons and frameshift mutations results in a genome amplification-defective phenotype (16). However, deletion of CP codons 2 to 189 has no effect on amplification, indicating that neither the CP-coding sequence up to codon 189 nor the product encoded by this sequence is required for amplification. Second, a cis-active RNA sequence between CP codons 211 and 246 (TEV nucleotides 9148 to 9252) (Fig. ​(Fig.1B)1B) is absolutely required, regardless of whether or not it is translated (Fig. ​(Fig.1B).1B). This cis-active sequence occupies a discrete internal region within the CP-coding sequence situated 243 to 347 nucleotides from the 3′ terminal poly(A) tail (16). Computer-generated models of the cis-active CP-coding sequence suggested that this region forms a series of stem-loop structures involving RNA from both CP-coding and 3′ nontranslated sequences (16). The involvement of cis-active 5′- and 3′-proximal genome sequence in promoting RNA replication is a well-documented feature of positive-strand RNA viruses (for examples, see references 25 and 28). The necessity of cis-active internal genomic RNA sequences for replication is less well documented, although there are a number of examples of such sequences in genomic and defective-interfering RNAs (1, 6, 8, 12, 16). Relatively little is known about the roles of RNA sequences and structures within the 3′ nontranslated region (NTR) of the potyvirus genome, as functional elements have yet to be identified. Rodriguez-Cerezo et al. (22) showed that a duplication mutation that resulted in lengthening of a proposed 3′ NTR stem in tobacco vein mottling potyvirus RNA caused an attenuated symptom phenotype, but the level of RNA accumulation in infected tissue was not affected. The basis for the attenuated phenotype is not clear. To further investigate the function of the CP-coding cis-active sequence and the 3′ NTR, as well as the proposed secondary structure throughout the region encompassing these sequences, a linker-scanning mutational analysis was done. Recombinant TEV genomes containing linker-scanning substitution mutations spanning most of the 3′-terminal 350 nucleotides were constructed, and their amplification activities in protoplasts were measured. In addition, several genomes with compensatory mutations to restore predicted secondary structures disrupted by the linker-scanning mutations were analyzed. The data support a model in which several secondary structures involving both CP-coding and 3′ NTR sequences are necessary for TEV RNA replication.