Deletion and Substitution Analysis Defines Regions and Residues within the Phosphoprotein of Bovine Respiratory Syncytial Virus That Affect Transcription, RNA Replication, and Interaction with the Nucleoprotein

Khattar, Sunil K.; Yunus, Abdul S.; Collins, Peter L.; Samal, Siba K.

doi:10.1006/viro.2001.0960

Cited by 20 publications

(11 citation statements)

References 42 publications

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“…The P protein is a homotetramer which interacts with N (Garcia-Barreno et al, 1996), L (Khattar et al, 2001), and M2-1 (Asenjo et al, 2006) and which is an essential co-factor of the polymerase. Although much shorter than other Paramyxoviridae counterparts, RSV P has similar activities.…”

Section: Virus Rnas and Proteinsmentioning

confidence: 99%

Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years

2011

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Human respiratory syncytial virus (RSV) is a ubiquitous pathogen that infects everyone worldwide early in life and is a leading cause of severe lower respiratory tract disease in the pediatric population as well as in the elderly and in profoundly immunosuppressed individuals. RSV is an enveloped, nonsegmented negative-sense RNA virus that is classified in Family Paramyxoviridae and is one of its more complex members. Although the replicative cycle of RSV follows the general pattern of the Paramyxoviridae, it encodes additional proteins. Two of these (NS1 and NS2) inhibit the host type I and type III interferon (IFN) responses, among other functions, and another gene encodes two novel RNA synthesis factors (M2-1 and M2-2). The attachment (G) glycoprotein also exhibits unusual features, such as high sequence variability, extensive glycosylation, cytokine mimicry, and a shed form that helps the virus evade neutralizing antibodies. RSV is notable for being able to efficiently infect early in life, with the peak of hospitalization at 2–3 months of age. It also is notable for the ability to reinfect symptomatically throughout life without need for significant antigenic change, although immunity from prior infection reduces disease. It is widely thought that re-infection is due to an ability of RSV to inhibit or subvert the host immune response. Mechanisms of viral pathogenesis remain controversial. RSV is notable for a historic, tragic pediatric vaccine failure involving a formalin-inactivated virus preparation that was evaluated in the 1960’s and that was poorly protective and paradoxically primed for enhanced RSV disease. RSV also is notable for the development of a successful strategy for passive immunoprophylaxis of high-risk infants using RSV-neutralizing antibodies. Vaccines and new antiviral drugs are in pre-clinical and clinical development, but controlling RSV remains a formidable challenge.

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Section: Virus Rnas and Proteinsmentioning

confidence: 99%

Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years

2011

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“…Studies of the P protein of bovine RSV, a homologue of human RSV, also showed that the C-terminal end and an internal region between residues 161 to 180 are required for N-P complex formation by coimmunoprecipitation analysis (30,28). Moreover, deletion studies of the bovine RSV P protein shows that only residues 41 to 80 could be removed without significantly impairing bovine RSV minigenome replication (27).…”

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confidence: 98%

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

Lü¹,

Brazas²,

Ma³

et al. 2002

J Virol

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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.Human respiratory syncytial virus (RSV) is the prototype virus in the Pneumovirus genus of the Paramyxoviridae family. The virus genome consists of a single 15-kb negative-stranded RNA that encodes 11 proteins (for a recent review, see reference 9). The nucleocapsid protein (N), phosphoprotein (P), and large polymerase protein (L) constitute the minimal components for viral RNA replication and transcription in vitro (16,43). The N protein associates with the genomic RNA to form the nucleocapsid, which serves as the template for RNA synthesis. The L protein is a multifunctional protein that contains RNA-dependent RNA polymerase catalytic motifs and is also probably responsible for capping and polyadenylation of viral mRNAs. However, the ...

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“…The RNP complex isolated from RSV-infected cells has been shown to possess an RNAdependent RNA polymerase (RdRp), mRNA polyadenylation, and mRNA capping activities in vitro (4,5,27). Multiple protein-protein and protein-RNA interactions mediate the assembly and activity of this complex (12,13,16,20,21,26,33,38,41). Thus, the RSV RNP is a complex that is rich in potential antiviral targets.…”

mentioning

confidence: 99%

Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase

Liuzzi¹,

Mason²,

Cartier³

et al. 2005

J Virol

119

106

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Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

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Deletion and Substitution Analysis Defines Regions and Residues within the Phosphoprotein of Bovine Respiratory Syncytial Virus That Affect Transcription, RNA Replication, and Interaction with the Nucleoprotein

Cited by 20 publications

References 42 publications

Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years

Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years

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

Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase

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