Using cell-free reactions, we investigated the role of the 5' cloverleaf (5'CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop 'b' and the C-rich sequence was inhibited. Taken together, these findings suggest that PCBP binding to the C-rich sequence was sufficient to support RNA stability and negative-strand synthesis. Mutational analysis of the three conserved structural elements in stem-loop 'd' showed that they were required for efficient negative- and positive-strand synthesis. Finally, we showed an RNA with a 5' terminal deletion (Delta49TD RNA), which was previously isolated from persistently infected cells, replicated at low but detectable levels in these reactions. Importantly, the critical replication elements identified in this study are still present in the Delta49TD RNA.
The precise relationship between the length of the 3' poly(A) tail and the replication and infectivity of poliovirus RNA was examined in this study. With both poly(A)(11) and poly(A)(12) RNAs, negative-strand synthesis was 1-3% of the level observed with poly(A)(80) RNA. In contrast, increasing the length of the poly(A) tail from (A)(12) to (A)(13) resulted in about a ten-fold increase in negative-strand synthesis. This increase continued with each successive increase in poly(A) tail length. With poly(A)(20) RNA, RNA synthesis approached the level observed with poly(A)(80) RNA. A similar relationship was observed between poly(A) tail length and the infectivity of the viral RNA. A replication model is described which suggests that viral RNA replication is dependent on a poly(A) tail that is long enough to bind poly(A) binding protein and to act as a template for VPg uridylylation and negative-strand initiation.
Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.
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