scholarly journals Casein Kinase 2-Mediated Phosphorylation of Respiratory Syncytial Virus Phosphoprotein P Is Essential for the Transcription Elongation Activity of the Viral Polymerase; Phosphorylation by Casein Kinase 1 Occurs Mainly at Ser215 and Is without Effect

1999 ◽  
Vol 73 (10) ◽  
pp. 8384-8392 ◽  
Author(s):  
Lesley C. Dupuy ◽  
Sean Dobson ◽  
Vira Bitko ◽  
Sailen Barik
Keyword(s):  
Respiratory Syncytial Virus ◽  
Ex Vivo ◽  
Transcription Elongation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Temperature Sensitive ◽  
Casein Kinase 1 ◽  
P Protein ◽  
Syncytial Virus

ABSTRACT The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser232 in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser232 was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser215. In vitro, P protein could be phosphorylated by purified CK1 at Ser215 but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser215, Ser232, and Ser237 were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.

scholarly journals Identification of Temperature-Sensitive Mutations in the Phosphoprotein of Respiratory Syncytial Virus That Are Likely Involved in Its Interaction with the Nucleoprotein

2002 ◽  
Vol 76 (6) ◽  
pp. 2871-2880 ◽  
Author(s):  
Bin Lu ◽  
Robert Brazas ◽  
Chien-Hui Ma ◽  
Tina Kristoff ◽  
Xing Cheng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Protein Function ◽  
Temperature Sensitive ◽  
P Gene ◽  
P Protein ◽  
Charged Residues ◽  
Cotton Rats ◽  
Syncytial Virus

ABSTRACT The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37°C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39°C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33°C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37°C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37°C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.

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 The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

2002 ◽  
Vol 76 (21) ◽  
pp. 10776-10784 ◽  
Author(s):  
Bin Lu ◽  
Chien-Hui Ma ◽  
Robert Brazas ◽  
Hong Jin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Virus Replication ◽  
Vero Cells ◽  
Phosphorylation Sites ◽  
N Protein ◽  
P Protein ◽  
Infected Cells ◽  
Syncytial Virus

ABSTRACT The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

scholarly journals Replacement of the F and G Proteins of Respiratory Syncytial Virus (RSV) Subgroup A with Those of Subgroup B Generates Chimeric Live Attenuated RSV Subgroup B Vaccine Candidates

1999 ◽  
Vol 73 (12) ◽  
pp. 9773-9780 ◽  
Author(s):  
S. S. Whitehead ◽  
M. G. Hill ◽  
C. Y. Firestone ◽  
M. St. Claire ◽  
W. R. Elkins ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Reverse Genetics ◽  
Gene Replacement ◽  
Chimeric Virus ◽  
Antigenic Determinants ◽  
Temperature Sensitive ◽  
Protective Antigens ◽  
Syncytial Virus ◽  
High Level

ABSTRACT Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create awt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. Thewt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

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 Respiratory Syncytial Virus Fusion Protein Mediates Inhibition of Mitogen-Induced T-Cell Proliferation by Contact

2002 ◽  
Vol 76 (3) ◽  
pp. 1163-1170 ◽  
Author(s):  
Jörg Schlender ◽  
Gunther Walliser ◽  
Jens Fricke ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Cell Contact ◽  
Activation Markers ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (HRSV) and bovine respiratory syncytial virus (BRSV) are major pathogens in infants and calves, respectively. Experimental BRSV infection of calves and lambs is associated with lymphopenia and a reduction in responsiveness of peripheral blood lymphocytes (PBLs) to mitogens ex vivo. In this report, we show that in vitro mitogen-induced proliferation of PBLs is inhibited after contact with RSV-infected and UV-inactivated cells or with cells expressing RSV envelope proteins on the cell surface. The protein responsible was identified as the RSV fusion protein (F), as cells infected with a recombinant RSV expressing F as the single envelope protein or cells transfected with a plasmid encoding F were able to induce this effect. Thus, direct contact with RSV F is necessary and sufficient to inhibit proliferation of PBLs. Interestingly, F derived from HRSV was more efficient in inhibiting human PBL proliferation, while F from BRSV was more efficient in inhibiting bovine PBLs. Since various T-cell activation markers were upregulated after presenter cell contact, T lymphocytes are viable and may still be activated by mitogen. However, a significant fraction of PBLs were delayed or defective in G0/G1 to S-phase transit.

scholarly journals Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

2021 ◽  
Author(s):  
Xiao-Dong Fang ◽  
Qiang Gao ◽  
Ying Zang ◽  
Ji-Hui Qiao ◽  
Dong-Min Gao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Virus Infection ◽  
Casein Kinase ◽  
Casein Kinase 1 ◽  
N Protein ◽  
Intrinsically Disordered ◽  
P Protein ◽  
L Proteins

Liquid–liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.

scholarly journals DEAD-box RNA Helicases Act as Nucleotide Exchange Factors for Casein Kinase 2

2021 ◽  
Author(s):  
Edoardo Fatti ◽  
Alexander Hirth ◽  
Andrea Svorinic ◽  
Matthias Guenther ◽  
Cristina-Maria Cruciat ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Substrate Inhibition ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Nucleotide Exchange ◽  
Rna Helicases ◽  
Casein Kinase 1

DDX RNA helicases promote RNA processing but DDX3X is also known to activate casein kinase 1 ϵ (CK1ϵ). Here we show that not only is protein kinase stimulation a latent property of other DDX proteins towards CK1ϵ, but that this extends to casein kinase 2 (CK2α2) as well. CK2α2 enzymatic activity is stimulated by a variety of DDX proteins and we identify DDX1/24/41/54 as physiological activators required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X reveals that CK1 and CK2 kinase stimulation engages its RNA binding- but not catalytic motifs. Mathematical modelling of enzyme kinetics and stopped-flow spectroscopy converge that DDX proteins function as nucleotide exchange factor towards CK2α2 that reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as a new principle in kinase regulation and an evolved function of DDX proteins.

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