Oligomerization of Mumps Virus Phosphoprotein

Abstract

The mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (P(N)), the oligomerization domain (P(O)), and the C-terminal domain (P(C)). While P(N) is known to relax the NP-bound RNA genome, the roles of P(O) and P(C) are not clear. In this study, we investigated the roles of P(O) and P(C) in viral RNA synthesis using mutational analysis and a minigenome system. We found that P(N) and P(C) functions can be trans-complemented. However, this complementation requires P(O), indicating that P(O) is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (P(N) to P(N) and P(C) to P(C)). Furthermore, we found that residues R231, K238, K253, and K260 in P(O) are critical for P's functions. We identified P(C) to be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.

Importance: MuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented in trans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

FIG 1

Schematic representation showing deletion mutants of MuV P. The amino acid residues for P_N, P_O, and P_C are provided. Mutant names correspond to the domains included in the P deletion mutants, and approximate sizes are provided. The terminal Flag tag locations are included for each mutant. NTD, N-terminal domain; OD, oligomerization domain; CTD, C-terminal domain.

FIG 2

trans-Complementation of P in minigenome system. (A) Minigenome activity of P, P_NO, and P_OC. Increasing amounts of P or P deletion mutants were transfected together with other plasmids as described in Materials and Methods. For P_NO-P_OC, the amount of each deletion mutant transfected was one-half of the total amount of P transfected. Renilla luciferase was the reporter gene in the minigenome, and firefly luciferase expression was used as a transfection control. The minigenome activity was measured and normalized as the ratio of Renilla luciferase activity to firefly luciferase activity (relative luciferase activity). (C) Minigenome activity of P, P_N, P_NO, and P_OC. Increasing amounts of P or P deletion mutants were used. P values were calculated using Student's t test. Error bars represent the SEMs of data from six replicates. (B and D) Immunoblotting was performed to detect the expression levels of NP, P, and the P deletion mutants. Lanes NC, control.

FIG 3

Mutation analysis of cysteine residue 356 of P. Residue C356 in full-length P and P_OC was mutated to alanine, serine, methionine, and valine and tested in the minigenome system. Increasing amounts of P or P deletion mutants were transfected together with other plasmids as described in Materials and Methods. The amounts transfected are provided in each graph. P values were calculated using Student's t test. Error bars represent the SEMs of data from six replicates.

FIG 4

P mutants with engineered cysteine residues dimerize in parallel orientation with biological activity. (A) Pairs of parallel chains are shown in green or blue. Side chains of mutated amino acids 213 (red) and 273 (yellow) are highlighted. The amino acid 213 residue not contained in the file was superimposed from the adjacent parallel chain. The figure was generated using the PyMOL program (43). (B) Schematic representation of incorporated cysteine residues for disulfide bond engineering. The sizes of the dimers in the parallel and antiparallel orientations are provided. (C) Assessment of the parallel oligomerization status of P deletion mutants containing cysteine residues under reducing and nonreducing conditions. (D) Minigenome activity of engineered P deletion mutants. Increasing amounts of P mutants were transfected with other plasmids as described in Materials and Methods. P values were calculated using Student's t test. Error bars represent the SEMs of data from six replicates. n.s., not significant.

FIG 5

P mutants with engineered cysteine residues for cross-linking of antiparallel helices. (A and B) Pairs of antiparallel chains are shown in green or blue. The kink at Gly246 located on only one pair of helices is highlighted. (B) Residues with side chains in close proximity are highlighted. These residues were mutated to cysteine to engineer disulfide bonds between antiparallel α helices in chains containing and lacking the Gly246 kink. The figure was generated using the PyMOL program (43). (C and D) Assessment of the antiparallel oligomerization status of P deletion mutants containing single (C) or double (D) cysteine residues under reducing and nonreducing conditions. (E) Iodoacetamide treatment (25 mM) to achieve the natural dimerization orientation of P mutants with engineered parallel or antiparallel disulfide bonds. (F) Minigenome activity of engineered P mutants. Increasing amounts of P mutants were transfected with other plasmids as described in Materials and Methods. P values were calculated using Student's t test. Error bars represent the SEMs of data from six replicates.

FIG 6

Charged residues in the P oligomerization domain are critical for P activity in the minigenome system. (A) Residues found in charged zippers were mutated to alanine and tested in the minigenome system. Increasing amounts of P mutants were transfected with other plasmids as described in Materials and Methods. P values were calculated using Student's t test. Error bars represent the SEMs of data from six replicates. (B) Immunoblotting was performed to detect the expression levels of NP, P, and the P mutants. NC, negative control (no P).

FIG 7

The C-terminal domain of P binds to L. (A) A schematic representation of the predicted amino acid residues for PIV5 P_N, P_O, and P_C from sequence analysis with MuV P is provided. Mutant names correspond to the domains included in the P deletion mutants. The terminal Flag tag locations as well as additional methionine residues (mmm), used to aid with visualization by radioactive labeling with ³⁵S, are included for each mutant. (B) Interaction between P domains and L_dI-III in 293T cells. Cells were transfected with full-length P, P chimeras, or L_dI-III or cotransfected with P and L_dI-III and labeled with ³⁵S. Cell lysates were precleared with Sepharose G beads and coimmunoprecipitated with monoclonal anti-MuV P (full-length MuV P), anti-PIV5 P (full-length PIV5 P), anti-Flag (chimeric P truncations), or anti-HA (L_dI-III) antibody and resolved on a 12.5% SDS-polyacrylamide gel.