scholarly journals Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase

2021 ◽  
Vol 17 (10) ◽  
pp. e1010002
Author(s):  
Simone Bach ◽  
Jana-Christin Demper ◽  
Paul Klemm ◽  
Julia Schlereth ◽  
Marcus Lechner ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Length Variation ◽  
Virus Rna ◽  
Promoter Recognition ◽  
Ebov Infection ◽  
Vesicular Stomatitis ◽  
Transcription Complexes

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.

Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis

Cell ◽  
1985 ◽  
Vol 41 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Heinz Arnheiter ◽  
Nancy L. Davis ◽  
Gail Wertz ◽  
Manfred Schubert ◽  
Robert A. Lazzarini
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Virus Rna ◽  
Vesicular Stomatitis

Role of ATP and protein kinases in vesicular stomatitis virus RNA synthesis

1988 ◽  
Vol 11 ◽  
pp. 29
Author(s):  
William B. Helfman ◽  
J.David Beckes ◽  
Lisa C. Childers ◽  
Jacques Perrault
Keyword(s):  
Protein Kinases ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Virus Rna ◽  
Vesicular Stomatitis

scholarly journals The role of ATP in in vitro vaccinia virus RNA synthesis effects of AMP-PNP and ATP gamma S.

1980 ◽  
Vol 255 (11) ◽  
pp. 5396-5403
Author(s):  
S. Shuman ◽  
E. Spencer ◽  
H. Furneaux ◽  
J. Hurwitz
Keyword(s):  
Vaccinia Virus ◽  
Rna Synthesis ◽  
Virus Rna ◽  
Gamma S

DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis

1990 ◽  
Vol 10 (7) ◽  
pp. 3415-3420
Author(s):  
M W Van Dyke ◽  
M Sawadogo
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Small Region ◽  
Protein Product ◽  
Differential Sensitivity ◽  
Yeast Cells ◽  
Promoter Regions ◽  
Regulatory Processes ◽  
Promoter Recognition ◽  
Transcription Complexes

The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.

scholarly journals The role of the priming loop in influenza A virus RNA synthesis

2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Aartjan J. W. te Velthuis ◽  
Nicole C. Robb ◽  
Achillefs N. Kapanidis ◽  
Ervin Fodor
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Virus Rna

scholarly journals Structures of the Mononegavirales Polymerases

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Bo Liang
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Mrnas ◽  
Future Directions ◽  
Virus Family ◽  
Viral Genomes ◽  
Vesicular Stomatitis ◽  
Syncytial Virus ◽  
Human Parainfluenza Virus ◽  
Negative Sense

ABSTRACT Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5′ methylated cap and 3′ poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales. We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales. We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

scholarly journals Binding of cellular p32 protein to the rubella virus P150 replicase protein via PxxPxR motifs

2012 ◽  
Vol 93 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Suganthi Suppiah ◽  
Heather A. Mousa ◽  
Wen-Pin Tzeng ◽  
Jason D. Matthews ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Rna Synthesis ◽  
Specific Binding ◽  
Infectious Clone ◽  
Binding Motifs ◽  
Replicase Protein ◽  
Virus Rna ◽  
Infected Cells ◽  
Proline Rich Region

A proline-rich region (PRR) within the rubella virus (RUBV) P150 replicase protein that contains three SH3 domain-binding motifs (PxxPxR) was investigated for its ability to bind cell proteins. Pull-down experiments using a glutathione S-transferase–PRR fusion revealed PxxPxR motif-specific binding with human p32 protein (gC1qR), which could be mediated by either of the first two motifs. This finding was of interest because p32 protein also binds to the RUBV capsid protein. Binding of p32 to P150 was confirmed and was abolished by mutation of the first two motifs. When mutations in the first two motifs were introduced into a RUBV cDNA infectious clone, virus replication was significantly impaired. However, virus RNA synthesis was found to be unaffected, and subsequent immunofluorescence analysis of RUBV-infected cells revealed co-localization of p32 and P150 but little overlap of p32 with RNA replication complexes, indicating that p32 does not participate directly in virus RNA synthesis. Thus, the role of p32 in RUBV replication remains unresolved.

scholarly journals An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors

2014 ◽  
Vol 95 (12) ◽  
pp. 2683-2692 ◽  
Author(s):  
Irina C. Albulescu ◽  
Ali Tas ◽  
Florine E. M. Scholte ◽  
Eric J. Snijder ◽  
Martijn J. van Hemert
Keyword(s):  
Mode Of Action ◽  
Chikungunya Virus ◽  
Rna Synthesis ◽  
Mechanistic Studies ◽  
Vitro Assay ◽  
Virus Rna ◽  
Infected Cells ◽  
Transcription Complexes ◽  
Crude Membrane Fraction

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

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