scholarly journals Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus

2000 ◽  
Vol 74 (13) ◽  
pp. 6006-6014 ◽  
Author(s):  
Rachel Fearns ◽  
Peter L. Collins ◽  
Mark E. Peeples
Keyword(s):  
Respiratory Syncytial Virus ◽  
Transcription Initiation ◽  
Rna Replication ◽  
Inhibitory Effect ◽  
Direct Transcription ◽  
Fourfold Increase ◽  
Syncytial Virus ◽  
High Degree ◽  
Pairing Potential ◽  
Transcription And Replication

ABSTRACT The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3′ ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3′ genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3′-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3′ end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancingcis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3′ end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3′ and 5′ ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.

scholarly journals Mapping the Transcription and Replication Promoters of Respiratory Syncytial Virus

2002 ◽  
Vol 76 (4) ◽  
pp. 1663-1672 ◽  
Author(s):  
Rachel Fearns ◽  
Mark E. Peeples ◽  
Peter L. Collins
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Replication ◽  
Saturation Mutagenesis ◽  
Common Element ◽  
Independent Events ◽  
Naturally Occurring ◽  
A Genome ◽  
Syncytial Virus ◽  
Overlapping Sets ◽  
Transcription And Replication

ABSTRACT An important, unresolved issue in mononegavirus biology is whether or not transcription is initiated by the same promoter as RNA replication. In this study, residues important for respiratory syncytial virus (RSV) transcription and RNA replication were identified by subjecting the first 26 nucleotides of genome RNA to saturation mutagenesis. This analysis was performed using a genome analog that allowed transcription and RNA replication to be dissociated from each other and monitored as independent events in an intracellular assay. This analysis showed that nucleotides 3C, 5C, 8U, 9U, 10U, and 11U were important for transcription and RNA replication. Additional nucleotides (1U, 2G, 6U, and 7U) were important for RNA replication, but not transcription. At position 4, G versus C or U augmented transcription and decreased replication, showing that the naturally occurring assignments in the genomic (4G) and antigenomic (4U) promoters are optimal for transcription and RNA replication, respectively. These data show that RSV transcription and RNA replication each involve a cis-acting signal at the very 3" end of the genome. This signal appears to contain a minimum, common element that functions in both transcription and RNA replication, defined by those substitutions that had similar effects on the two processes. Apart from these common nucleotides, other positions were involved in RNA replication but not transcription or had different effects on the two processes. This indicates that the promoters for transcription and replication involve overlapping sets of nucleotides at the very 3" end of the genome and provides evidence that the nucleotide preferences for the two processes are not identical.

scholarly journals Effect of Zinc Salts on Respiratory Syncytial Virus Replication

2004 ◽  
Vol 48 (3) ◽  
pp. 783-790 ◽  
Author(s):  
Rahaman O. Suara ◽  
James E. Crowe
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Zinc Acetate ◽  
Lower Respiratory Tract ◽  
Divalent Cations ◽  
Cell Monolayer ◽  
Inhibitory Effect ◽  
Plaque Formation ◽  
Zinc Salts ◽  
Syncytial Virus

ABSTRACT Zinc supplementation decreases the morbidity of lower respiratory tract infection in pediatric patients in the developing world. We sought to determine if zinc mediates a specific inhibitory effect against the major cause of pediatric lower respiratory tract disease, respiratory syncytial virus (RSV). We determined the in vitro inhibitory effect of three zinc salts (zinc acetate, lactate, and sulfate) on the replication of RSV at various concentrations of 10 and 1 mM and 100 and 10 μM. The degree of inhibition of RSV replication was examined in the presence of zinc during preincubation, adsorption, or penetration and was compared with that caused by salts of other divalent cations. Complete inhibition of RSV plaque formation was observed at 1 and 10 mM, representing reductions that were ≥106-fold. At the lowest concentration tested, 10 μM, we observed ≥1,000-fold reductions in RSV yield when zinc was present during preincubation, adsorption, penetration, or egress of virus. The therapeutic indices, determined as ratios of 50% toxicity concentration to 50% inhibitory concentration, were 100, 150, and 120 for zinc acetate, zinc lactate, and zinc sulfate, respectively. The inhibitory effect of zinc salts on RSV was concentration dependent and was not observed with other salts containing divalent cations such as calcium, magnesium, and manganese. RSV plaque formation was prevented by pretreatment of HEp-2 cell monolayer cultures with zinc or by addition of zinc to methylcellulose overlay media after infection. The results of this study suggest that zinc mediates antiviral activity on RSV by altering the ability of the cell to support RSV replication.

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 Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion

2010 ◽  
Vol 107 (22) ◽  
pp. 10226-10231 ◽  
Author(s):  
S. L. Noton ◽  
V. M. Cowton ◽  
C. R. Zack ◽  
D. R. McGivern ◽  
R. Fearns
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Replication ◽  
Syncytial Virus

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication

2021 ◽  
Author(s):  
Ashley C. Beavis ◽  
Kim C. Tran ◽  
Enrico R. Barrozo ◽  
Shannon I. Phan ◽  
Michael N. Teng ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Viral Rna ◽  
Growth Defect ◽  
Compensatory Mutation ◽  
Phosphorylation Sites ◽  
Viral Growth ◽  
P Protein ◽  
Genome Transcription ◽  
Syncytial Virus ◽  
Transcription And Replication

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, RNA virus in the family Pneumoviridae and genus Orthopneumoviridae that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all serine S and T residues to alanine (A) and first analyzed their effect on genome transcription and replication using a minigenome system. We found that the mutation of eight residues resulted in significantly reduced minigenome activity compared to wild-type P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (T151A, S156A, T160A, and S23A), suggesting RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four rescued, rRSV-T160A caused a minor growth defect compared to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication, and S156 plays a critical role in viral RNA synthesis. Importance RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylate these residues can lead to kinase inhibitors and anti-viral drugs for this important human pathogen.

scholarly journals Suppression of the Induction of Alpha, Beta, and Gamma Interferons by the NS1 and NS2 Proteins of Human Respiratory Syncytial Virus in Human Epithelial Cells and Macrophages

2004 ◽  
Vol 78 (8) ◽  
pp. 4363-4369 ◽  
Author(s):  
Kirsten M. Spann ◽  
Kim-C. Tran ◽  
Bo Chi ◽  
Ronald L. Rabin ◽  
Peter L. Collins
Keyword(s):  
Respiratory Syncytial Virus ◽  
Epithelial Cells ◽  
Human Peripheral Blood ◽  
Inhibitory Effect ◽  
Human Respiratory Syncytial Virus ◽  
Blood Monocytes ◽  
Pulmonary Epithelial Cells ◽  
Alpha Beta ◽  
Syncytial Virus ◽  
The Individual

ABSTRACT Wild-type human respiratory syncytial virus (HRSV) is a poor inducer of alpha/beta interferons (IFN-α/β). However, recombinant HRSV lacking the NS1 and NS2 genes (ΔNS1/2) induced high levels of IFN-α and -β in human pulmonary epithelial cells (A549) as well as in macrophages derived from primary human peripheral blood monocytes. Results with NS1 and NS2 single- and double-gene-deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role. The relative contributions of the individual NS proteins were the converse of that recently described for bovine RSV (J. F. Valarcher, J. Furze, S. Wyld, R. Cook, K. K. Conzelmann, and G. Taylor, J. Virol. 77:8426-8439, 2003). This pattern of inhibition by HRSV NS1 and NS2 also extended to the newly described antiviral cytokines IFN-λ1, -2 and -3.

scholarly journals Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections

2005 ◽  
Vol 79 (16) ◽  
pp. 10190-10199 ◽  
Author(s):  
Antonieta Guerrero-Plata ◽  
Samuel Baron ◽  
Joyce S. Poast ◽  
Patrick A. Adegboyega ◽  
Antonella Casola ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Respiratory Infections ◽  
Significant Degree ◽  
Inhibitory Effect ◽  
Human Metapneumovirus ◽  
Absolute Amount ◽  
Potent Inducer ◽  
Cpg Oligodeoxynucleotides ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the Paramyxoviridae family, Pneumovirinae subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-α/β) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-α in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-α, or directly with recombinant IFN-α showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-α than hMPV. Similarly, when the ability to directly induce IFN-α production was assessed, RSV was clearly a weaker inducer of IFN-α than hMPV, as shown by both kinetics and the absolute amount of IFN-α secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-α production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-α production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-α in the lung during infection by members of the Pneumovirinae family.

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