Molecular studies of poliovirus: Inhibition of picornavirus and hepatitis C virus IRES-mediated translation and a molecular dissection of polioviral 2A(pro) proteinase

My collaborators and I undertook a novel approach using oligonucleotides to inhibit translation mediated by the picornaviral and hepatitis C virus (HCV) IRES elements. The mechanism for inhibition of translation by these oligonucleotides is likely to be binding of trans-acting factor(s) from the translational apparatus.; Eleven oligodeoxynucleotides were chosen that are complementary to plus-sense RNA of poliovirus type I (Mahoney) (PV1(M)) 5′-NTR. The inhibitory effects of these oligonucleotides on the cap-dependent translation were examined with rabbit globin mRNA.; Genetic data has demonstrated that poliovirus 2Apro plays multiple functions in viral proliferation (protein processing, RNA replication and translational activation). It is not known whether the multiple functions of 2Apro are due to independent functional domains or to domains involved in proteolysis. There are no biochemical data available that indicate a direct role for 2Apro in viral genome replication. We conducted a molecular dissection on the coding sequence for poliovirus 2Apro . In this study, a functional domain in the C-terminus of 2A pro was identified by genetic analyses. The C-terminal sequences of poliovirus 2Apro is EAMEQ-NH2. Rhinovirus type II (HRV2) 2A pro lacks this domain in its C-terminal sequence and, indeed, the HRV2 2Apro cannot substitute for poliovirus to yield a viable virus. Deletion of the EEAME sequence from poliovirus 2Apro is lethal without significantly influencing proteinase function. On the other hand, the addition of EAME to HRV2 2Apro, yielding a C-terminus of this enzyme of EEEAMEQ, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. PV1(M) 2A pro could be provided in trans to rescue the lethal deletion of EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C-terminus of poliovirus 2Apro is involved in particle formation. We conclude that the C-terminus of poliovirus 2Apro is an essential domain for viral RNA replication, but it is not essential for proteolytic processing. (Abstract shortened by UMI.)...