Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone.
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.
Fujimura
Tsutomu
T
Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Avda. del Campo Charro s/n Salamanca 37007, Spain. tfujimura@usal.es
Solórzano
Alicia
A
Esteban
Rosa
R
D002457:Cell Extracts; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012328:RNA Viruses / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000821:virology*; D014779:Virus Replication
D002457:Cell Extracts; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases