scholarly journals Identification of the Bunyamwera bunyavirus transcription termination signal

2006 ◽  
Vol 87 (1) ◽  
pp. 189-198 ◽  
Author(s):  
John N. Barr ◽  
John W. Rodgers ◽  
Gail W. Wertz
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Transcription Termination ◽  
Single Species ◽  
Negative Strand ◽  
The Family ◽  
L Segment ◽  
Bunyamwera Virus ◽  
Genomic Template

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3′ ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5′ terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3′-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5′ non-translated region (NTR) of the S segment. A 6 nt region (3′-GUCGAC-5′) within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3′-UGUCG-5′, that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5′ NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.

scholarly journals Bunyamwera Bunyavirus RNA Synthesis Requires Cooperation of 3′- and 5′-Terminal Sequences

2004 ◽  
Vol 78 (3) ◽  
pp. 1129-1138 ◽  
Author(s):  
John N. Barr ◽  
Gail W. Wertz
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Sequence Conservation ◽  
Functional Requirement ◽  
Base Pairing ◽  
Replication Assay ◽  
Perfect Complementarity ◽  
Negative Sense ◽  
Bunyamwera Virus

ABSTRACT Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3′- and 5′-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3′ and 5′ NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3′- and 5′-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3′ and 5′ NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3′ and 5′ NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3′ and 5′ NTRs were selected.

scholarly journals Role of the Conserved Nucleotide Mismatch within 3′- and 5′-Terminal Regions of Bunyamwera Virus in Signaling Transcription

2005 ◽  
Vol 79 (6) ◽  
pp. 3586-3594 ◽  
Author(s):  
John N. Barr ◽  
Gail W. Wertz
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Nucleotide Identity ◽  
Nucleotide Position ◽  
Transcription Activity ◽  
Nucleotide Mismatch ◽  
Negative Sense ◽  
Bunyamwera Virus ◽  
Transcription And Replication

ABSTRACT Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3′ and 5′ nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable. Here, we investigate the basis for the fundamentally different signaling abilities of genomic and antigenomic strands. We show that the identity of only nucleotide position 9 within the genomic 3′ NTR is critical for the different RNA synthesis characteristics of genomic and antigenomic strands, thus identifying this nucleotide as an essential component of the transcription promoter. This nucleotide is distinctive, as it interrupts an unbroken run of conserved complementary nucleotides within the 3′ and 5′ NTRs of all three segments. Our results show that the conserved mismatched arrangement of this nucleotide plays no detectable role in signaling transcription. Instead, we show that the transcription-signaling ability of this position is entirely dependent on its nucleotide identity. We further show that while a U residue at 3′ position 9 is strongly preferred for transcription activity in the context of the genomic promoter, it does not signal transcription in the context of the antigenomic promoter. Therefore, our results show that the identity of 3′ position 9 is crucial for signaling BUNV transcription; however, it is not the sole determinant.

Role of GS-5734 (Remdesivir) in inhibiting SARS-CoV and MERS-CoV: The expected role of GS-5734 (Remdesivir) in COVID-19 (2019-nCoV) - VYTR hypothesis.

2020 ◽  
Vol 11 (SPL1) ◽  
Author(s):  
Vityala Yethindra
Keyword(s):  
Clinical Trials ◽  
Antiviral Activity ◽  
Protease Inhibitors ◽  
Human Body ◽  
Broad Spectrum ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Improved Outcomes ◽  
The Family

Coronaviruses (CoVs) are enveloped RNA viruses related to the family Coronaviridae, the order Nirdovales, and observed in humans and other mammals. In December 2019, many pneumonia cases reported by patients with unknown causes, mainly associated with seafood and wet animal market in Wuhan, China, and where clinically resembled viral pneumonia. At present, there is no existence of antiviral drugs for the treatment of CoV infections. The results of our study are GS-5734 strongly inhibits SARS-CoV and MERS-CoV in HAE cells, GS-5734 inhibits CoVs at early stages in replication by inhibiting viral RNA synthesis, the absence of ExoN-mediated proofreading in viruses sensitive to treatment with GS-5734. Protease inhibitors can show improved outcomes in some coronaviruses, but mostly 99% of protease inhibitors bind to proteins present in the human body, and only 1% attacks on existed viruses. The expected role of GS-5734 (Remdesivir) in the 2019-nCoV - VYTR hypothesis explained. As broad-spectrum drugs are capable of inhibiting CoV infections, GS-5734 is a broad-spectrum drug and may show inhibition on CoV infections and 2019-nCoV. GS-5734 will show desired results regarding antiviral activity against 2019-nCoV as it showed potent antiviral activity in other CoVs. More clinical trials and experiments needed to prove that GS-5734 (Remdesivir) is a potential and effective drug to treat 2019-nCoV.

scholarly journals Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Codon Usage ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Genomic Rna ◽  
Negative Strand ◽  
Unique Mechanism

ABSTRACTNegative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. Byin situanalyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCENegative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

scholarly journals A case for a reverse-frame coding sequence in a group of positive-sense RNA viruses

2019 ◽  
Author(s):  
Adam M. Dinan ◽  
Nina I. Lukhovitskaya ◽  
Ingrida Olendraite ◽  
Andrew E. Firth
Keyword(s):  
De Novo ◽  
Rna Viruses ◽  
Gene Evolution ◽  
Stop Codon ◽  
Messenger Rnas ◽  
Negative Strand ◽  
Public Data ◽  
Wide Range ◽  
The Family ◽  
Positive Sense

ABSTRACTPositive-sense single-stranded RNA viruses form the largest and most diverse group of eukaryote-infecting viruses. Their genomes comprise one or more segments of coding-sense RNA that function directly as messenger RNAs upon release into the cytoplasm of infected cells. Positive-sense RNA viruses are generally accepted to encode proteins solely on the positive strand. However, we previously identified a surprisingly long (~1000 codons) open reading frame (ORF) on the negative strand of some members of the familyNarnaviridaewhich, together with RNA bacteriophages of the familyLeviviridae, form a sister group to all other positive-sense RNA viruses. Here, we completed the genomes of three mosquito-associated narnaviruses, all of which have the long reverse-frame ORF. We systematically identified narnaviral sequences in public data sets from a wide range of sources, including arthropod, fungi and plant transcriptomic datasets. Long reverse-frame ORFs are widespread in one clade of narnaviruses, where they frequently occupy >95% of the genome. The reverse-frame ORFs correspond to a specific avoidance of CUA, UUA and UCA codons (i.e. stop codon reverse complements) in the forward-frame RNA-dependent RNA polymerase ORF. However, absence of these codons cannot be explained by other factors such as inability to decode these codons or GC3 bias. Together with other analyses, we provide the strongest evidence yet of coding capacity on the negative strand of a positive-sense RNA virus. As these ORFs comprise some of the longest known overlapping genes, their study may be of broad relevance to understanding overlapping gene evolution andde novoorigin of genes.

scholarly journals Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

2021 ◽  
Vol 17 (12) ◽  
pp. e1010151
Author(s):  
Afzaal M. Shareef ◽  
Barbara Ludeke ◽  
Paul Jordan ◽  
Jerome Deval ◽  
Rachel Fearns
Keyword(s):  
Rna Synthesis ◽  
De Novo ◽  
Rna Viruses ◽  
Rna Virus ◽  
Promoter Sequence ◽  
Structural Features ◽  
Marburg Virus ◽  
Initiation Mechanism ◽  
Negative Strand ◽  
Syncytial Virus

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

scholarly journals Vesicular Stomatitis Viruses Resistant to the Methylase Inhibitor Sinefungin Upregulate RNA Synthesis and Reveal Mutations That Affect mRNA Cap Methylation

2007 ◽  
Vol 81 (8) ◽  
pp. 4104-4115 ◽  
Author(s):  
Jianrong Li ◽  
John S. Chorba ◽  
Sean P. J. Whelan
Keyword(s):  
Gene Expression ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Gene ◽  
Negative Strand ◽  
L Protein ◽  
Conserved Sequence ◽  
Viral Yield ◽  
Vesicular Stomatitis

ABSTRACT Sinefungin (SIN), a natural S-adenosyl-l-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2′-O (2′-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2′-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.

scholarly journals Asymmetric Trimeric Ring Structure of the Nucleocapsid Protein of Tospovirus

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Keisuke Komoda ◽  
Masanori Narita ◽  
Keitaro Yamashita ◽  
Isao Tanaka ◽  
Min Yao
Keyword(s):  
Life Cycle ◽  
Nucleic Acids ◽  
Rna Viruses ◽  
Economic Damage ◽  
Genomic Rnas ◽  
Content Type ◽  
Negative Strand ◽  
N Protein ◽  
The Family ◽  
N Proteins

ABSTRACT Tomato spotted wilt virus (TSWV), belonging to the genus Tospovirus of the family Bunyaviridae, causes significant economic damage to several vegetables and ornamental plants worldwide. Similar to those of all other negative-strand RNA viruses, the nucleocapsid (N) protein plays very important roles in its viral life cycle. N proteins protect genomic RNAs by encapsidation and form a viral ribonucleoprotein complex (vRNP) with some RNA-dependent RNA polymerases. Here we show the crystal structure of the N protein from TSWV. Protomers of TSWV N proteins consist of three parts: the N arm, C arm, and core domain. Unlike N proteins of other negative-strand RNA viruses, the TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, the N and C arms of the N protein interact with the core domains of two adjacent N proteins. By solving the crystal structures of the TSWV N protein with nucleic acids, we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. These characteristics are similar to those of N proteins of other viruses of the family Bunyaviridae. Based on these observations, we discuss possibilities of a TSWV encapsidation model. IMPORTANCE Tospoviruses cause significant crop losses throughout the world. Particularly, TSWV has an extremely wide host range (>1,000 plant species, including dicots and monocots), and worldwide losses are estimated to be in excess of $1 billion annually. Despite such importance, no proteins of tospoviruses have been elucidated so far. Among TSWV-encoded proteins, the N protein is required for assembling the viral genomic RNA into the viral ribonucleoprotein (vRNP), which is involved in various steps of the life cycle of these viruses, such as RNA replication, virus particle formation, and cell-to-cell movement. This study revealed the structure of the N protein, with or without nucleic acids, of TSWV as the first virus of the genus Tospovirus, so it completed our view of the N proteins of the family Bunyaviridae.

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