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.

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 In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

2019 ◽  
Vol 295 (3) ◽  
pp. 883-895 ◽  
Author(s):  
Yunrong Gao ◽  
Dongdong Cao ◽  
Hyunjun Max Ahn ◽  
Anshuman Swain ◽  
Shaylan Hill ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Synthesis ◽  
Genomic Rnas ◽  
Viral Rnas ◽  
N Protein ◽  
Syncytial Virus ◽  
Nucleoprotein N ◽  
Negative Sense ◽  
Transcription And Replication

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

scholarly journals Hsp90 is required for snakehead vesiculovirus replication via stabilizing the viral L protein

2021 ◽  
Author(s):  
Yanwei Zhang ◽  
Yong-An Zhang ◽  
Jiagang Tu
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Insoluble Fraction ◽  
Viral Rna ◽  
Cellular Proteins ◽  
Economic Losses ◽  
L Protein ◽  
Hsp90 Activity ◽  
The Stability

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. The Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensure the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. Importance It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help understand the polymerase function of negative-stranded RNA viruses.

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 Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 126
Author(s):  
Justin M. Su ◽  
Maxwell Z. Wilson ◽  
Charles E. Samuel ◽  
Dzwokai Ma
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Host Responses ◽  
Future Research ◽  
Virus Infections ◽  
Infected Cells ◽  
And Function ◽  
Negative Sense ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented negative-sense (NNS) RNA viruses have uncovered a key role of LLPS in the formation of viral inclusion bodies (IBs), sites of viral protein concentration in the cytoplasm of infected cells. These studies further reveal the structural and functional complexity of viral IB factories and provide a foundation for their future research. Herein, we review the literature leading to the discovery of LLPS-driven formation of IBs in NNS RNA virus-infected cells and the identification of viral scaffold components involved, and then outline important questions and challenges for IB assembly and disassembly. We discuss the functional implications of LLPS in the life cycle of NNS RNA viruses and host responses to infection. Finally, we speculate on the potential mechanisms underlying IB maturation, a phenomenon relevant to many human diseases.

scholarly journals Overlapping Signals for Transcription and Replication at the 3′ Terminus of the Vesicular Stomatitis Virus Genome

1999 ◽  
Vol 73 (1) ◽  
pp. 444-452 ◽  
Author(s):  
Tong Li ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Virus Genome ◽  
Gene Analysis ◽  
Genomic Rna ◽  
Wild Type ◽  
Transcription Activity ◽  
Transcription Reinitiation ◽  
Vesicular Stomatitis ◽  
Negative Sense ◽  
Transcription And Replication

ABSTRACT Transcription and replication signals within the negative-sense genomic RNA of vesicular stomatitis virus (VSV) are located at the 3′ terminus. To identify these signals, we have used a transcription- and replication-competent minigenome of VSV to generate a series of deletions spanning the first 47 nucleotides at the 3′ terminus of the VSV genome corresponding to the leader gene. Analysis of these mutants for their ability to replicate showed that deletion of sequences within the first 24 nucleotides abrogated or greatly reduced the level of replication. Deletion of downstream sequences from nucleotides 25 to 47 reduced the level of replication only to 55 to 70% of that of the parental template. When transcription activity of these templates was measured, the first 24 nucleotides were also found to be required for transcription, since deletion of these sequences blocked or significantly reduced transcription. Downstream sequences from nucleotides 25 to 47 were necessary for optimal levels of transcription. Furthermore, replacement of sequences within the 25 to 47 nucleotides with random heterologous nonviral sequences generated mutant templates that replicated well (65 to 70% of the wild-type levels) but were transcribed poorly (10 to 15% of the wild-type levels). These results suggest that the minimal promoter for transcription and replication could be as small as the first 19 nucleotides and is contained within the 3′-terminal 24 nucleotides of the VSV genome. The sequences from nucleotides 25 to 47 may play a more important role in optimal transcription than in replication. Our results also show that deletion of sequences within the leader gene does not influence the site of transcription reinitiation of the downstream gene.

scholarly journals Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates

2004 ◽  
Vol 78 (17) ◽  
pp. 9568-9572 ◽  
Author(s):  
Frank T. Vreede ◽  
Tanis E. Jung ◽  
George G. Brownlee
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Mrna Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Replicative Intermediates ◽  
Negative Sense ◽  
Transcription And Replication

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a “switching” mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.

scholarly journals Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

2021 ◽  
Author(s):  
Jingru Fang ◽  
Colette Pietzsch ◽  
George Tsaprailis ◽  
Gogce Crynen ◽  
Kelvin Frank Cho ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Host Factors ◽  
Lassa Virus ◽  
High Confidence ◽  
Biotin Ligase ◽  
Infected Cells ◽  
New Biology ◽  
Negative Sense ◽  
Transcription And Replication

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the negative-sense RNA viral genome in the cytoplasm of infected cells. We hypothesized that interactions with an array of cellular proteins may enable LASV polymerase to execute distinct viral RNA biosynthetic processes. To investigate this hypothesis, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate viral transcription and replication. We engineered a LASV polymerase-biotin ligase TurboID fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed us to identify 42 high-confidence hits that interact with LASV polymerase. We performed an siRNA screen to evaluate the role of the identified interactors in LASV infection, which uncovered six host factors for which their depletion affected LASV infection. We found that one polymerase interactor, eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), physically and functionally associated with LASV polymerase, exhibiting proviral activity. Accordingly, pharmacological targeting of GSPT1 resulted in strong inhibition of LASV infection. In summary, our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactomes, which can reveal new biology and uncover novel targets for the development of antivirals against LASV.

scholarly journals Mutagenic Analysis of Hazara Nairovirus Nontranslated Regions during Single- and Multistep Growth Identifies both Attenuating and Functionally Critical Sequences for Virus Replication

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Daniele F. Mega ◽  
Jack Fuller ◽  
Beatriz Álvarez-Rodríguez ◽  
Jamel Mankouri ◽  
Roger Hewson ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Adjacent Segment ◽  
Virus Growth ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Multiplication Cycle ◽  
Negative Sense

ABSTRACT Hazara nairovirus (HAZV) is a member of the family Nairoviridae in the order Bunyavirales and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3′ and 5′ NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3′ NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5′ NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent cis-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed. IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3′ and 5′ ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3′ and 5′ NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

scholarly journals The structure of a native orthobunyavirus ribonucleoprotein reveals a key role for viral RNA in maintaining its helical architecture

2021 ◽  
Author(s):  
Francis Hopkins ◽  
Beatriz Alvarez-Rodriguez ◽  
George Heath ◽  
Kyriakoulla Panayi ◽  
Samantha Hover ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Longitudinal Axis ◽  
Emerging Pathogens ◽  
Cryo Electron Microscopy ◽  
Therapeutic Measures ◽  
Human Use ◽  
Negative Sense ◽  
Bunyamwera Virus

The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active template for RNA synthesis and the form in which the genome is packaged into virions, functions that require inherent flexibility. We present a pseudo-atomic model of a native RNP purified from Bunyamwera virus (BUNV), the prototypical Bunyavirales member, based on a cryo-electron microscopy (cryo-EM) average at 13 A resolution with subsequent fitting of the BUNV NP crystal structure by molecular dynamics. We show the BUNV RNP possesses relaxed helical architecture, with successive helical turns separated by ~18 A. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms, with no helix-stabilizing interactions along the longitudinal axis. Instead, EM analysis of RNase-treated RNPs suggests their chain integrity is dependent on the encapsidated genomic RNA, thus providing the molecular basis for RNP flexibility. Overall, this work will assist in designing anti-viral compounds targeting the RNP and inform studies on bunyaviral RNP assembly, packaging and RNA replication.

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