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.
The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.
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