A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

Abstract

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frameshifting by a mechanism indistinguishable from that of retroviruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.The pol genes of retroviruses are expressed as gag-pol or gag-pro-pol fusion polyproteins (1) formed either by in-frame read-through of termination codons (2, 3) or by ribosomal frameshifting (4-6). Both mechanisms allow for production of multiple proteins from a single, unmodified mRNA.In Rous sarcoma virus (RSV), gag and pol genes overlap, with pol being in the -1 frame with respect to gag (7). In 5% of translations, a -1 frameshifting event allows ribosomes to miss the gag termination codon and continue to translate the pol gene, producing a gag-pol fusion protein (8, 9). A -1 ribosomal frameshifting has also been described in coronaviruses [(+) single-stranded (ss) RNA genomes] (10, 11), phage T7 (12), and in the dnaX gene of Escherichia coli (13)(14)(15). A +1 ribosomal frameshift is seen in the yeast retrotransposon Tyl (16)(17)(18) and in the E. coli release factor 2 (19-21).The signals responsible for -1 ribosomal frameshifting include a "slippery site" heptamer, X XXY YYZ (gag reading frame indicated; X = A, U, or G; Y = A or U; Z = A, U, or C), followed by a stem-loop structure that can be involved in an RNA pseudoknot (4,9,13,20,22,23). A pseudoknot is base pairing of the loop with a sequence 3' of a stem-loop (24, 25). The "simultaneous slippage" model of Jacks et al. (4) proposes that the tRNAs bound at the ribosomal P site to XXY and at the A site to YYZ simultaneously slip back 1 base on the mRNA to pair with XXX and YYY, respectively. Because their nonwobble bases remain properly paired, this can happen at a finite rate (Fig. 1). The stem-loop structure has been demonstrated to be essential for efficient frameshifting in RSV (4), infectious bronchitis virus (23), and the E. coli dnaX gene (13) and is predicted to occur following the slippery site heptamers of a number of other retroviruses (4, 9, 23). RNA secondary structure downstream of the slippery site may slow or stall ribosomes such that they remain in the slippery site longer, thus promoting frameshifting (4).The L-A g...

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous

“…This is in accordance with the synthesis of the viral polymerase as a fusion with CP by a -1 ribosomal frameshifting, event that occur at low frequency [3]. XdV-L2 can be considered a replicative intermediate of XdV-L1A, because it is identical in sequence but without 989 bp at the 5 0 -end.…”

“…Its dsRNA genome of 4,579 bp in length has two ORFs, the 5 0 ORF encodes for the major capsid protein (CP) (Gag or CP, 76-81 kDa) and the 3 0 ORF for the RNA-dependent RNA polymerase (Pol or RdRp). Both ORFs are overlapped in 16-130 nt and the viral polymerase is expressed as a fusion CP-Pol protein (predicted size of 170 kDa) generated by a ribosomal-1 frameshifting [3][4][5]. Some strains of S. cerevisiae have an additional dsRNA molecule of 1.6-1.8 kbp called M-dsRNA, that encoded fort a killer toxin and self-immunity; this ''satellite virus'' depends on the viral proteins encoded by L-A (the ''helper virus'') for its encapsidation and replication [6].…”

Functional analysis of the uL11 protein impact on translational machinery

“…Noteworthy, the -1 PRF is strategically applied by the L-A virus for propagation. 50 As shown in our previous report, the lack of P1 and P2 proteins on the ribosome, which form the lateral stalk component of the GAC, was associated with increased L-A virus replication. Interestingly, this was accompanied with increased eEF1A association with the GAC region, 51 indicating a functional interplay between P1/P2, uL11, and eEF1A during the ribosomal decoding step.…”

5S rRNA is involved in fidelity of translational reading frame

“…The mopmutation was identified by the dark blue phenotype of a single colony (EMS56). EMS56 was cured of p-1 and was retransformed with p-1 or PO (previously refered to as pTI25) (DINMAN et al 1991, DINMAN and WICKNER 1992. From Pgalactosidase activities with each plasmid the efficiency of -1 ribosomal frameshifting was 5.7% in EMS56 and 1.9% in unmutagenized cells, a ratio of 3.0.…”

“…Plasmids: Plasmids pTI25 and pF8 have been described (DINMAN et al 1991). Briefly, pTI25 is the 0-frame control plasmid, while pF8 is the -1 ribosomal frameshift tester.…”