Reticulocyte RNA-dependent RNA polymerase

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.     

RNA-dependent RNA polymerase of tobacco plants

Several properties of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) active fractions obtained from tobacco plants, be they uninfected, infected with tobacco mosaic virus, or infected with tobacco necrosis virus, were compared. By the seven criteria tested, the RNA-dependent RNA polymerase from these three sources behaved the same, although its activity is greatly but variably stimulated by the two virus infections. It thus appears probable that these two viruses do not code for this enzyme, but rely for their replication on their ability to stimulate production of a host enzyme. The conclusion that cells contain RNA replicating capability represents a modification of the central dogma of molecular biology.     

Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: −17, −14, −13, −11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.     

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.     

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.     

Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli.

The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase). The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol. To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli. The plasmids consisted of fusions between the E. coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides. E. coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).oligo(U) poly(U) polymerase assay. The purified RNA polymerase activity from E. coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein. The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.     

Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase

Positive-strand RNA viruses include a large number of human and animal pathogens whose essential RNA-dependent RNA polymerases (RdRPs) share a structurally homologous core with an encircled active site. RdRPs are targets for antiviral drug development, but these efforts are hindered by limited structural information about the RdRP catalytic cycle. To further our understanding of RdRP function, we assembled, purified, and then crystallized poliovirus elongation complexes after multiple rounds of nucleotide incorporation. Here we present structures capturing the active polymerase and its nucleotide triphosphate complexes in four distinct states, leading us to propose a six-state catalytic cycle involving residues that are highly conserved among positive-strand RNA virus RdRPs. The structures indicate that RdRPs use a fully prepositioned templating base for nucleotide recognition and close their active sites for catalysis using a novel structural rearrangement in the palm domain. The data also suggest that translocation by RDRPs may not be directly linked to the conformational changes responsible for active site closure and reopening.     

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.     

RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains.

The L-A double-stranded RNA virus of Saccharomyces cerevisiae makes a gag-pol fusion protein by a -1 ribosomal frameshift. The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants. We have carried out "alanine-scanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N (. = any amino acid) and GDD. By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely defined the extent of domains and specific residues essential for viral replication. Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely beta-sheet structure.     

Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase.

Three lines of evidence indicate that RNA-dependent RNA synthesis occurs in mouse erythroleukemia cells. The first involves labeling studies with [3H]uridine and shows a greater initial labeling rate of globin RNA in the cytoplasm than in the nucleus. Labeled globin RNA found in the cytoplasm after a very short pulse with tritiated uridine is of the "mature" 9S size while labeled globin RNA in the nuclei is exclusively in the form of 15S precursor molecules, suggesting that cytoplasmic globin RNA is not of nuclear origin. A high concentration of actinomycin D has no effect on the initial rate of labeling of cytoplasmic globin RNA, supporting this conclusion. Other experiments showed that the labeling of cytoplasmic globin RNA does not involve end addition to preexisting globin RNA. The second line of evidence is the identification of globin RNA minus strand in the cytoplasm of differentiated murine erythroleukemia cells by hybridization with single-stranded DNA probes containing the strand of the same sense as globin mRNA. This material has the same electrophoretic mobility as globin RNA and hybridizes with probes containing only the 5' part or only the 3' part of the gene suggesting that it is a full size copy of globin RNA. Finally, in murine erythroleukemia cells an RNA-dependent RNA polymerase activity is detected by using poly(A) . oligo(U) as a template-primer combination. This activity increases significantly after induction, suggesting that it is differentiation specific.