scholarly journals In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Dongdong Cao ◽  
Yunrong Gao ◽  
Claire Roesler ◽  
Samantha Rice ◽  
Paul D'Cunha ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fine Mapping ◽  
Rna Synthesis ◽  
De Novo ◽  
Rna Viruses ◽  
Rna Virus ◽  
Promoter Sequence ◽  
Mechanistic Understanding ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis. IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

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 Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase

Virology ◽  
2014 ◽  
Vol 462-463 ◽  
pp. 318-327 ◽  
Author(s):  
Sarah L. Noton ◽  
Waleed Aljabr ◽  
Julian A. Hiscox ◽  
David A. Matthews ◽  
Rachel Fearns
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Factors Affecting ◽  
Syncytial Virus

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 4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication

2021 ◽  
Author(s):  
Julien Sourimant ◽  
Carolin Lieber ◽  
Megha Aggarwal ◽  
Robert Cox ◽  
Josef Wolf ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Broad Spectrum ◽  
Rna Virus ◽  
Oral Treatment ◽  
The Elderly ◽  
Virus Infections ◽  
Once Daily ◽  
Syncytial Virus ◽  
Well Differentiated

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections.

scholarly journals Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

2021 ◽  
Vol 118 (13) ◽  
pp. e2020969118
Author(s):  
Cyril Le Nouën ◽  
Thomas McCarty ◽  
Lijuan Yang ◽  
Michael Brown ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
De Novo ◽  
Point Mutations ◽  
Surface Glycoprotein ◽  
Temperature Sensitive ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Codon Pair ◽  
Syncytial Virus

Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs—called codon-pair deoptimization (CPD)—provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B–infected cells were encapsidated, expressed high levels of F, and complemented Min B replicationin trans. This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common “defective interfering particles” that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

scholarly journals Protein-Primed and De Novo Initiation of RNA Synthesis by Norovirus 3Dpol

2006 ◽  
Vol 80 (14) ◽  
pp. 7060-7069 ◽  
Author(s):  
Jacques Rohayem ◽  
Ivonne Robel ◽  
Katrin Jäger ◽  
Ulrike Scheffler ◽  
Wolfram Rudolph
Keyword(s):  
Rna Synthesis ◽  
De Novo ◽  
Rna Viruses ◽  
Rna Dependent Rna Polymerase ◽  
Polyadenylated Rna ◽  
Initiation Of Replication ◽  
Replication Strategy ◽  
High Concentrations ◽  
Novel Model

ABSTRACT Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3Dpol), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3Dpol was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3Dpol generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3′ termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3Dpol initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3Dpol initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3Dpol, with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.

scholarly journals Optimization of the Codon Pair Usage of Human Respiratory Syncytial Virus Paradoxically Resulted in Reduced Viral Replication In Vivo and Reduced Immunogenicity

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Cyril Le Nouën ◽  
Cindy L. Luongo ◽  
Lijuan Yang ◽  
Steffen Mueller ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Viral Replication ◽  
Virus Replication ◽  
Neutralizing Antibodies ◽  
Rna Virus ◽  
Humoral Immune ◽  
Codon Pair ◽  
Syncytial Virus ◽  
Codon Pairs

ABSTRACT We subjected various open reading frames (ORFs) in the genome of respiratory syncytial virus (RSV) to codon pair optimization (CPO) by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (Δ1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro. All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response. IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon pair optimized (CPO) by increasing the content of codon pairs that are overrepresented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response.

scholarly journals Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

2001 ◽  
Vol 75 (23) ◽  
pp. 11328-11335 ◽  
Author(s):  
Roderick S. Tang ◽  
Nick Nguyen ◽  
Xing Cheng ◽  
Hong Jin
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Protein Function ◽  
Rna Synthesis ◽  
Human Respiratory Syncytial Virus ◽  
Wild Type ◽  
C Terminus ◽  
Cotton Rats ◽  
Syncytial Virus

ABSTRACT The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys3-His1motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using β-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys3-His1motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys3-His1 motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys3-His1 motif. This indicates that the Cys3-His1 motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length β-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Inhibitory Effect of Sargassum fusiforme and Its Components on Replication of Respiratory Syncytial Virus In Vitro and In Vivo

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 548
Author(s):  
Kiramage Chathuranga ◽  
Asela Weerawardhana ◽  
Niranjan Dodantenna ◽  
Lakmal Ranathunga ◽  
Won-Kyung Cho ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Agent ◽  
Active Components ◽  
Sargassum Fusiforme ◽  
Antiviral Effects ◽  
Improve Life Expectancy ◽  
Rsv Infection ◽  
Syncytial Virus

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

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