Native Replication Intermediates of the Yeast 20 S RNA Virus Have a Single-stranded RNA Backbone

Rosa Esteban,Alicia Solórzano,Tsutomu Fujimura

doi:10.1074/jbc.m412048200

Rosa Esteban, Alicia Solórzano + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m412048200

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Abstract

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Highlights

Positive strand RNA viruses encode their RNA-dependent RNA polymerases (RdRps)1 and utilize them to replicate their RNA genomes in conjunction with other viral or host proteins
Radioactive nucleotides were first incorporated into the doublestranded RNA (dsRNA) form with a peak at 20 min, and the label in the dsRNA form decreased gradually (Fig. 1B)
During the first 5-min labeling period, most of labeled nucleotides were incorporated into the dsRNA form

Summary

The abbreviations used are

RdRp, RNA-dependent RNA polymerase; ssRNA; single-stranded RNA; dsRNA, double-stranded RNA; nt, nucleotide(s); Cs, cytidines; RI, replicative intermediate. The viral genome is small (2514 nt) and has no 3Ј-poly(A) tail and perhaps no 5Ј-cap structure [8] It encodes only a single protein of 91 kDa (p91) [9, 10]. Because the viral genomes do not encode coat proteins, the RNA genomes are not encapsidated into viral particles (14 –16) Instead, they form ribonucleoprotein complexes with their cognate RNA polymerases in a 1:1 stoichiometry and reside in the host cytoplasm [17]. We analyzed replication intermediates of 20 S RNA synthesis in vitro and found that the native replication intermediates have a single-stranded RNA (ssRNA) backbone, consisting of a positive strand less than unit length and a template negative strand loosely associated, perhaps held by p91.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION