A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA

摘要

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT–PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3′ elongation of mRNA with guanosine residues, thus immediately preserving the 3′ end of the RNA and creating a unique poly(A)–oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC9T6) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT–PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.