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.

Regulation of the primary expression of the early adenovirus transcription units.

Abstract: The time course of appearance of transcriptional activity from five early adenovirus type 2 transcription units has been determined. RNA complementary to region 1A (1-4.4 map units), the first region to be transcribed, was detectable at 45 min after infection; a maximal rate of RNA synthesis was reached at 3 h after infection and was maintained thereafter for at least 6 h. RNA from region 2 (75-56 map units), which encodes the mRNA for the 72,000-dalton DNA-binding protein, was the last to be synthesized; transcription commenced at about 2 h postinfection, reached a maximum at 7 h, and then declined. Transcription of regions 3 (76-86 map units) and 4 (99-91 map units) reached a maximal value at 3 h postinfection. The rates of RNA synthesis from these regions then declined over the next 6 h. The decline of transcription from regions 2 and 4 appeared to be a specific repression of these transcription units. The repression did not occur in the absence of protein synthesis, suggesting that a viral protein might be involved. Transcription of all early regions was initiated and continued for at least 2 to 3 h in cells that were treated with cycloheximide or emetine before and during infection, suggesting that at least the initiation of RNA synthesis from the five early adenovirus type 2 transcription units does not depend on the formation of a viral protein. Moreover, mRNA was formed in the absence of protein synthesis that hybridized to DNA fragments representing each of the five early transcription units. The increase in mRNA accumulation in the presence of cycloheximide (or emetine) does not appear to be due to increased RNA synthesis; thus, either increased mRNA stability or increased efficiency of nuclear RNA processing must occur.

The coronavirus proofreading exoribonuclease mediates extensive viral recombination.

Abstract: Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.

Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes

Abstract: Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

Abstract: Hepatitis E virus (HEV) is an important etiological agent of acute epidemic and sporadic enteric hepatitis affecting millions of people mainly in developing countries. The first confirmed HEV epidemic, due to contamination of drinking water in New Delhi, India, was described in 1955. In addition to large epidemics in India and China, there are annually about 2 million sporadic cases of HEV infections in India alone. The mortality among HEV patients has been 0.5 to 4%, except in the case of pregnant women, for whom the average mortality is 20% (for reviews, see references 22 and 33). Recently, closely related viruses have been isolated from pigs, cows, sheep, goats, and rats, indicating zoonotic HEV infections (13). HEV is a nonenveloped virus with a diameter of 27 to 34 nm (9, 10), which does not replicate in cell cultures (1). The complete nucleotide sequence of the positive-strand RNA genome has been determined for several isolates from different parts of the world (40). (For references, see reference 8.) The HEV genome consists of a 27-nucleotide (nt)-long 5′ noncoding region followed by an open reading frame (ORF) coding for a nonstructural protein of 1,693 aa residues. ORF2 starts 38 nt downstream of the termination codon of ORF1 and codes for the capsid protein of 660 aa. ORF3 between nt 5105 and 5476 overlaps with ORF2 and codes for a 123-aa-long polypeptide with unknown function. The 3′ noncoding region is 65 nt, ending with a 150- to 200-nt-long poly(A) tail. A recent finding indicates that the HEV genome has an m7G cap structure at the 5′ end of the RNA (15). Expression of HEV capsid protein in COS-1 cells revealed that it is a glycoprotein with a size of 88 kDa, which is synthesized as a precursor (ggPORF2), processed to gPORF2 by cleavage of a signal sequence of 22 aa, and transported to the plasma membrane (14, 46). A more stable cytosolic form of capsid protein PORF2 with a size of 78 kDa has been detected in HepG2 cells by using a Semliki Forest virus (SFV) expression vector (41, 42). So far, which form of capsid protein is responsible for the production of infectious HEV is unknown. A truncated 50-kDa form of PORF2 produced in insect cells is able to assemble into hollow particles with an icosahedral symmetry of T=1 (25, 44). Expression of ORF3 in eukaryotic cells revealed that it is a phosphoprotein with affinity for the cytoskeleton through a hydrophobic amino-terminal domain of 32 aa residues (45). Expression of complete ORF1 coding for 1,693 aa residues in vitro, in Escherichia coli and in HepG2 cells, resulted in a large 186-kDa protein that was not autocatalytically processed (7). In another study, prolonged in vivo expression yielded N-terminal 78-kDa and C-terminal 107-kDa fragments (34). Transfection of in vitro-synthesized RNA, consisting of the complete HEV genome, to HepG2 cells resulted in synthesis of ORF1, ORF2, and ORF3 products as well as release of small amounts of infectious virus into the culture medium (29). However, no 186-kDa protein was detected in pulse-chase experiments. Instead, region-specific antisera precipitated smaller 35- to 40-kDa polypeptides. Computer-assisted assignments for the putative functions of HEV ORF1 suggested that the amino-terminal domain from 60 to 240 aa may be a methyltransferase followed by a Y domain with unknown function, a papain-like protease domain (around 440 to 610 aa), a proline-rich spacer region, an X domain of unknown function, a helicase domain (around 960 to 1,200 aa), and an RNA polymerase domain (around 1,200 to 1,700 aa) (17). None of the predicted functions has been experimentally verified. Indeed, it was recently shown that one of the putative protease active site residues is not conserved in different isolates and that mutation of the predicted active site cysteine did not abrogate P186 processing (34). Thus, the predicted protease appears not to exist or have enzymatic activity. Based on the ORF1 sequence, HEV belongs to a large alphavirus-like superfamily of positive-strand RNA viruses with putative methyltransferase, protease, helicase, and RNA polymerase domains (18). The distinctive methyltransferase domain is the hallmark of the alphavirus-like superfamily (28, 35). It contains sequence similarity to cellular S-adenosylmethionine (AdoMet)-dependent methyltransferases (5). In addition to guanine-7-methyltransferase activity (20, 36), the amino-terminal part of alphavirus nonstructural polyprotein, termed nsP1, also posesses guanylyltransferase activity (2). Both activities are needed in the capping of viral mRNAs (3, 5), and thus the conserved domain can more appropriately be designated as the capping enzyme domain. Both nsP1-catalyzed reactions are virus specific (i.e., there are no known host cell enzymes with similar specificities). Methyltransferase catalyzes the transfer of a methyl group from AdoMet to GTP, resulting in m7GTP, which forms the covalent complex nsP1-m7GMP. These reactions can be inhibited by cap analogs (22). In the present paper, we show the first enzymatic activity found for the HEV nonstructural protein. Truncated nonstructural protein P110 derived from HEV ORF1 produced in insect cells has virus-specific methyltransferase and guanylyltransferase activities similar to those of alphavirus replicase proteins. This finding provides a novel approach for development of specific inhibitors against hepatitis E infection.

Recombinant negative strand RNA virus expression systems

Abstract: Recombinant negative strand virus RNA templates which may be used to express heterologous gene products and/or to construct chimeric viruses are described. Influenza viral polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicative that the promoter lay solely within the 15 nucleotide 3' terminus. Sequences not specific for the influenza viral termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus sense cRNA were copied at low levels. The specificity for recognition of the virus-sense promoter was further defined by site-specific mutagenesis. It was also found that increased level of viral protein were required in order to catalyze both the cap-endonuclease primed and primer-free RNA synthesis from these model templates as well as from genomic length RNAs. This indicated that this reconstituted system had catalytic properties very similar to those of native viral RNPs. High levels of expression of a heterologous gene was obtained using the constructs and methods described. The system was exemplified using Influenza and respiratory syncytial virus.