Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

Abstract

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Figure 1

Infection of tobacco plants with polyadenylated AMV RNAs. Plants were inoculated with RNAs 1–3 with modified 3′ termini as indicated. A0, no modification; A40 and A80, extension with 40 and 80 A-residues, respectively; Xn, extension with plasmid-derived transcripts of 110 (RNAs 1 and 2) or 600 (RNA 3) nucleotides. Inocula plus or minus CP were used as indicated. Northern blots were loaded with RNA extracted from 200 μg (lanes 1–4) or 10 μg (lanes 5–8) of leaf material and hybridized with a probe detecting plus-strand RNAs 1–4. Relative sample sizes are indicated at the bottom, and the positions of RNAs 1–4 are indicated at the left of the blot.

Figure 2

Infection of tobacco protoplasts with mixtures of polyadenylated and nonpolyadenylated AMV RNAs. Protoplasts were inoculated with RNAs 1–3 with modified 3′ termini as indicated. 0, no modification; 80, extension with 80 A-residues. Inocula plus or minus CP were used as indicated. Northern blots were loaded with RNA extracted from 10,000 (lanes 1–8) or 500 (lane 9) protoplasts and hybridized with probes detecting plus-strand RNAs 1–4 (A) or minus-strand RNAs 1–3 (B). Relative sample sizes are indicated at the bottom, and the positions of plus-strand RNAs 1–4 and minus-strand RNAs 1–3 are indicated at the left of the blots.

Figure 3

Role of 5′-leader sequences and polyadenylation of RNAs 1 and 2 in the initiation of AMV RNA replication. Protoplasts were inoculated with RNAs 1–3 with modified 5′ or 3′ termini as indicated. 0, no modification; 80, 3′ extension with 80 A-residues; L4, 5′ UTR replaced by 5′ UTR of RNA 4; minus sign, no RNA 3 present in the inoculum. Inocula plus or minus CP were used as indicated. Northern blots were loaded with RNA extracted from 10,000 (lanes 1–7, 9, and 11–14) or 500 (lanes 8 and 10) protoplasts and hybridized with probes detecting plus-strand RNAs 1–4 (A) or minus-strand RNAs 1–3 (B). Relative sample sizes are indicated at the bottom, and the positions of plus-strand RNAs 1–4 and minus-strand RNAs 1–3 are indicated at the left of the blots.

Figure 4

Initiation of minus-strand RNA synthesis and enhancement of translation of RNA 4 in protoplasts by wt and mutant CP. (A–C) Protoplasts were inoculated with wt RNAs 1 and 2 (lane 1), wt RNAs 1 and 2 plus CP (lane 11), or wt RNAs 1 and 2 plus RNA 4 encoding wt CP (lanes 2 and 3), or the mutant CPs R17A, ΔN16, ΔN10, R25A, R16K17, and K13A (lanes 4–9, respectively). RNA 4 in the inoculum was capped (lanes 3–9) or not capped (lane 2). Northern blots were loaded with RNA extracted from 10,000 protoplasts and hybridized with probes detecting plus-strand RNAs 1–4 (A) or minus-strand RNAs 1–3 (B). The positions of plus-strand RNAs 1, 2, and 4 and minus-strand RNAs 1 and 2 are indicated at the left of the blots. (C) Analysis of CP translated from RNA 4 (lanes 2–9) or derived from the inoculum (lane 10). Protein was extracted from 10,000 protoplasts and analyzed by Western blotting with a CP antiserum. The position of wt CP is indicated at the left of the blot. (D) Translation in reticulocyte lysates of RNA 4 transcripts corresponding to the RNA 4 transcripts used in A–C. Synthesis of CP in the lysates was analyzed by Western blotting with a CP antiserum. The position of wt CP is indicated at the left of the blot.

Figure 5

CP stimulates translation of AMV RNAs in trans. Tobacco protoplasts were mock-inoculated (lane 1) or inoculated with RNA 4 transcripts encoding wt CP (lane 2), mutant CPs R17A (lane 3), R16K17 (lane 4), ΔN16 (lane 5), ΔN10 (lane 6), or mixtures of ΔN10 and R17A (lane 7), ΔN10 and R16K17 (lane 8), ΔN10 and wt CP (lane 9), and ΔN16 and wt CP (lane 10). Protein was extracted from 10,000 protoplasts and analyzed by Western blotting with a CP antiserum. The position of wt CP is indicated at the left of the blot.