Poliovirus infection enhances the formation of two ribonucleoprotein complexes at the 3' end of viral negative-strand RNA

Abstract

To identify proteins involved in the formation of replication complexes at the 3 end of poliovirus negativestrand RNA, a combined in vitro biochemical and in vivo genetic approach was used. Five subgenomic cDNA constructs were generated to transcribe different negative-strand RNA fragments. In UV cross-linking assays, distinct differences in binding of proteins in extracts from poliovirus-infected and uninfected cells to virusspecific, radiolabeled transcripts were observed. Two proteins present in extracts from poliovirus-infected cells with approximate molecular masses of 36 and 38 kDa were shown to cross-link to the 3 end of poliovirus negative-strand RNA. Appearance of the 36-and 38-kDa proteins in UV cross-linking assays can be detected 3 to 3.5 h after infection, and cross-linking reaches maximum levels by 5 h after infection. The binding site for the 36-kDa protein overlaps with the computer-predicted loop b region of stem-loop I, the so-called cloverleaf structure, and the RNA sequence of this region is required for efficient binding. Transfection of full-length, positive-sense RNA containing a five-nucleotide substitution (positions 20 to 25) in the loop b region of stem-loop I into tissue culture cells yielded only viral isolates with a reversion at position 24 (U3C). This finding demonstrates that the wild-type cytidine residue at position 24 is essential for virus replication. RNA binding studies with transcripts corresponding to the 3 end of negative-strand RNA suggest that complex formation with the 36-kDa protein plays an essential role during the viral life cycle.

Cis-acting RNA elements in human and animal plus-strand RNA viruses

“…Unexpectedly, it was observed that the PV 3′ NTR (2 stem loops) could be replaced by the 3′ NTRs of HRV14 (1 stem loop) or of coxsackievirus B4 (CVB4) (3 stem loops) [23]. Even more surprising was the finding that the PV or HRV14 3′ NTRs could be deleted without loss of viability in HeLa cells [31,32].…”

Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins

“…In addition to hnRNP A1, C, and K (Gustin and Sarnow, 2001), these include nucleolin, a primarily nuclear RNA-binding protein thought to be involved in rRNA metabolism, DNA replication, and apoptosis (Waggoner and Sarnow, 1998), Sam 68, an SH domain-binding protein (McBride et al, 1996), and the La autoantigen, a protein that binds to the poliovirus IRES (Meerovitch et al, 1993). We have previously described the interaction of hnRNP C with the 3′ end of poliovirus negative-strand RNA and with poliovirus RNA replication polypeptides (Roehl and Semler, 1995;Brunner et al, 2005). A mutated form of hnRNP C1, with a truncation of the protein-protein interaction auxiliary domain, impaired RNA synthesis in an in vitro replication assay.…”

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions

“…Our results suggest that while specific 3-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3 terminus of template RNAs within tight membranous replication complexes.The macromolecular interactions which direct the highly efficient and specific process of picornavirus RNA synthesis in the cytoplasm of an infected host cell remain elusive. Several recent reports pertaining to the mechanism of initiation of positive-strand RNA synthesis have significantly advanced the understanding of this RNA replication process (1,2,7,20,29,34,35); however, progress toward an understanding of negative-strand RNA synthesis has been lagging. The major cisacting molecular genetic determinant for picornavirus negative-strand RNA synthesis is believed to reside in the genomic RNA 3Ј noncoding region (3Ј NCR) immediately upstream of the genetically encoded polyadenylate tract (39,46,47).…”