Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer

2008 ◽  
Vol 35 (11) ◽  
pp. 1091 ◽  
Author(s):  
Lei Wang ◽  
Neil A. Smith ◽  
Lan Zhang ◽  
Elizabeth S. Dennis ◽  
Peter M. Waterhouse ◽  
...  

RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract.

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Huansheng Wu ◽  
Liuyuan Shi ◽  
Yina Zhang ◽  
Xiran Peng ◽  
Tuyuan Zheng ◽  
...  

ABSTRACTUbiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We report that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV).In vivodetection revealed the ubiquitination of VP1 protein in IBDV-infected target organs and different cells but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C terminus. The K751 ubiquitination is independent of VP1’s interaction with VP3 and eukaryotic initiation factor 4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C terminus of VP1 enhanced its polymerase activity. The K751-to-R mutation of VP1 protein did not block the rescue of IBDV but decreased the replication ability of IBDV. Our data demonstrate that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.IMPORTANCEAvibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression, and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C terminus of VP1 is the target site for ubiquitin, and its ubiquitination is independent of VP1’s interaction with VP3 and eukaryotic initiation factor 4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We conclude that VP1 is the ubiquitin-modified protein and reveal the mechanism by which VP1 promotes avibirnavirus replication.


2020 ◽  
Author(s):  
Jialei Sun

AbstractThe outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+ or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+ promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.


1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.


2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4322
Author(s):  
Luyun Huang ◽  
John Gledhill ◽  
Craig E. Cameron

2010 ◽  
Vol 107 (45) ◽  
pp. 19151-19156 ◽  
Author(s):  
C.-Y. Su ◽  
T.-J. R. Cheng ◽  
M.-I. Lin ◽  
S.-Y. Wang ◽  
W.-I. Huang ◽  
...  

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