Key result
A newly developed G-tailing RT-PCR method demonstrated that the poly(A) tail of hepatitis A virus RNA is elongated during the course of viral replication.
The development of a new G-tailing RT-PCR method allows for more accurate determination of poly(A) tail lengths in RNA viruses like HAV.
Supports investigation of poly(A) tail function in HAV; leaves open implications for human infection and treatment.
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(dC(9)T(6)) 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.
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Yuri Kusov (2001) studied Hepatitis A virus (HAV) RNA replication. G-tailing RT-PCR method was evaluated on Poly(A) tail length of viral RNA. A newly developed G-tailing RT-PCR method demonstrated that the poly(A) tail of hepatitis A virus RNA is elongated during the course of viral replication.
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