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

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.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 ...

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

“…Recently, we and the other groups have reported that other regions in P protein are also involved in its interaction with N protein (20,22). This report also demonstrated that phosphorylation of RSV P protein was important for N-P protein interaction.…”

“…The plasmids expressing RSV N, P, and L proteins under the control of the T7 promoter (in the pCITE vector) were described previously (18). The RSV minigenome, pRSVCAT, encodes a negative-sense chloramphenicol acetyltransferase (CAT) gene under the control of the T7 promoter (22). pRSVCAT/EGFP was constructed by inserting an enhanced green fluorescent protein (EGFP) gene, which was flanked by the RSV gene start and gene end sequence downstream of the CAT gene, into pRSVCAT.…”

“…For Western blotting, Vero cells were infected with each virus at an MOI of 1.0, and the cells were lysed in protein lysis buffer at 48 h postinfection. Detection of viral proteins in the blot by polyclonal anti-RSV antibody was performed as described by Lu et al (22).…”

Protein-Protein Interactions in RSV Assembly: Potential Targets for Attenuating RSV Strains

“…SH 0 is the 7.5 kDa non-glycosylated form and is the most common form expressed. SH g is the N-linked glycosylated form (15)(16)(17)(18)(19) kDa) which is a precursor to SH p ; the latter (21-40 kDa) is a polylactosaminoglycan modified form of the protein. SH t is a truncated, 4.8 kDa protein generated by translation initiation at in internal AUG [45].…”

“…The C-terminus of P is required for interaction with N [17]. Yeast two-hybrid analysis shows that the C-terminus of P is important in forming heterodimers with N [18], with changes in two residues in this region of P (E176 and G172) resulting in a temperature-sensitive phenotype due to reduced interaction with N [19]. Phosphorylation of P has been shown to be dispensable for most of its assembly functions, e.g., P-P, P-N and P-M2-1 interactions in cell-free and cell culture systems [20,21].…”