RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro

Abstract: After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Abstract: Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.

Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication

Abstract: Plus-strand RNA viruses replicate their genomes in infected cells by using a replicase complex comprised of viral and host proteins that assembles in association with cellular membranes (1, 4, 15). In infected cells, viral replicases are able to specifically recognize and selectively replicate their cognate viral RNAs from a heterogeneous pool of cellular RNA molecules. In contrast, in vitro studies have shown that many purified viral replicase complexes are able to utilize heterologous promoter or initiation elements quite efficiently (11, 28, 39). These conflicting findings between in vivo and in vitro data have led to models that attribute selective recognition of viral templates to host proteins (4, 13). Phage Qbeta utilizes this type of mechanism whereby the S1 ribosomal protein and elongation factor Tu in the four-subunit replicase complex mediate viral template recognition (3, 13). There is also evidence that virally encoded proteins can facilitate selective template recruitment to the viral replicase complex. Examples in this category include the 1a protein of Brome mosaic virus and the 126-kDa protein of Tomato mosaic virus (5, 17, 33). However, in both of these cases it is not known whether the respective viral RNAs are recognized directly by these replicase proteins or require assistance from host proteins (6). In Poliovirus, specific binding of 3CD protein to the 5′-terminal cloverleaf-like structure has been reported, and this interaction likely contributes to template selection into replication (9, 38). However, the host poly(C) binding protein 2 also interacts specifically with the same 5′-terminal RNA structure, and it, too, is proposed to be involved in mediating template selection (9, 38). In general, the contribution of viral and cellular proteins to RNA template recognition by cognate replicases is largely unknown in most viral systems. Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the large family Tombusviridae. Its genome encodes two proteins involved in viral RNA replication, the prereadthrough product p33 and the readthrough product p92 (Fig. 1A and B). Both of these proteins are essential for RNA replication (18, 22), they are part of the viral replicase complex (23), and they accumulate in vivo at a ratio of 20:1, respectively (23, 31). The less plentiful p92 functions as the viral RNA-dependent RNA polymerase (RdRp), while the role of the more abundant prereadthrough p33 is undefined (37). In other tombusviruses, the orthologues of TBSV p33 have been shown to be targeted to mitochondrial or peroxisomal membranes, the presumed sites of tombusvirus RNA replication (29). In TBSV, both p33 and p92 are membrane associated (23, 31). RNA-binding domains have also been identified in these proteins (22, 26), and the ability of p33 to interact with itself and p92 has been demonstrated (27). The cumulative data support an essential role for both TBSV p33 and p92 in viral RNA replication, with p92 comprising the catalytic subunit responsible for RNA synthesis and p33 playing a critical but unknown auxiliary role. FIG. 1. Specific binding of the recombinant p33 replicase protein to RII(+)-SL in vitro. (A) Schematic representation of the p33 and p92 replicase proteins of TBSV. The sequence of p33 is identical to the N-terminal overlapping (prereadthrough) domain ... In this paper we tested the binding of a recombinant TBSV p33 to four conserved regions of the viral genome known to affect replication (37). We demonstrate that p33 binds selectively in vitro to a conserved RNA motif within the p92 coding region of the viral genome. The specific recognition of this RNA element is dependent on a C · C mismatch positioned within a helix, and this key determinant of p33 binding is also essential for TBSV replication in host cells. We propose that this interaction directs viral template recruitment into replication and suggest that this mechanism may also apply to other members of the large family Tombusviridae.