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A region of mumps virus nucleoprotein affects defective interfering particle production
Jacquline Risalvato1, James Zengel1, Mark Phillips1
1Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA 30601, USA.
Abstract:
Mumps virus (MuV) is a negative-sense, single-stranded RNA virus belonging to the family Paramyxoviridae. MuV causes acute infection of the parotid glands, and the infection can result in severe cases of encephalitis, meningitis and deafness in humans. The non-segmented RNA genome of MuV is encapsidated by the nucleocapsid protein (NP), which forms the ribonucleoprotein (RNP) complex that serves as a template for viral RNA synthesis. To make viral genomic RNA accessible to the viral polymerase, a conformational change within NP occurs. Recently, an atomic model of the NP of MuV was developed with cryogenic-electron microscopy (cryo-EM) using PIV5 NP crystal structure as a homology template. To examine NP's structure and function, we performed mutational analysis of MuV NP at region(s) proposed to play a role in accessing viral RNA. The MuV NP mutants containing G185P, A197Q, Q200R and groups denoted as Top (N63G, P139D, A197Q), Tip (P109E, N121G, A124R) and Bottom (G21S, E29T, P43N, R93Q, R304Q) were first tested in a minigenome system. All mutations resulted in reduced reporter gene activities with Q200R and Bottom having the most severe negative effects. Rescuing of recombinant viruses with these mutations was attempted, and only MuV mutants '185 (G185P)', '197 (A197Q)' and 'Top (N63G, P139D, A197A)' were obtained. The 'Top' MuV mutant exhibited normal growth kinetics at low multiplicities of infection (MOIs); however, at high MOIs, the virus had reduced peak litres than low MOIs. Further analysis indicates that production of defective interfering particles (DI particles or DIPs) was enhanced by the mutant virus, indicating that this region, a known alpha-helix hinge region, is important for full-length genome replication, suggesting that it plays a role in maintaining stability of viral RNA-dependent RNA-polymerase on RNP template during MuV viral RNA synthesis. Understanding the production of DI particles will lead to a better understanding of MuV pathogenesis, as well as its replication/transcription process.
Insights
Mutating mumps virus (MuV) nucleocapsid protein (NP) regions impacts viral RNA synthesis and defective interfering particle (DI particle) production. Understanding these mutations aids in comprehending MuV replication and pathogenesis.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Mumps virus (MuV), a Paramyxoviridae family member, causes significant human illness, including encephalitis and deafness.
- The MuV nucleocapsid protein (NP) encapsidates the viral RNA genome, forming the ribonucleoprotein (RNP) complex essential for viral RNA synthesis.
- Accessing the viral RNA genome requires conformational changes in NP, a process not fully understood at the atomic level.
Purpose of the Study:
- To investigate the structural and functional roles of specific regions within the MuV nucleocapsid protein (NP).
- To analyze the impact of NP mutations on viral RNA synthesis and replication efficiency.
- To understand the relationship between NP mutations, defective interfering particle (DI particle) production, and MuV pathogenesis.
Main Methods:
- Cryo-electron microscopy (cryo-EM) was used to determine the atomic model of MuV NP, with PIV5 NP structure serving as a homology template.
- Site-directed mutagenesis was employed to create MuV NP mutants, including single point mutations (G185P, A197Q, Q200R) and group mutations (Top, Tip, Bottom).
- Minigenome systems and recombinant virus rescue were used to assess the functional impact of NP mutations on viral RNA synthesis and virus growth kinetics.
Main Results:
- Mutations in MuV NP generally reduced reporter gene activity in a minigenome system, with Q200R and the Bottom mutant showing the most severe effects.
- Only MuV mutants '185 (G185P)', '197 (A197Q)', and 'Top (N63G, P139D, A197A)' were successfully rescued.
- The 'Top' mutant exhibited altered growth kinetics at high multiplicities of infection (MOIs) and enhanced production of defective interfering particles (DI particles), indicating impaired full-length genome replication.
Conclusions:
- Specific regions of the MuV NP, particularly the alpha-helix hinge region targeted by the 'Top' mutation, are crucial for efficient full-length genome replication.
- These NP regions play a vital role in maintaining the stability of the viral RNA-dependent RNA-polymerase on the RNP template during MuV RNA synthesis.
- Enhanced DI particle production by the 'Top' mutant suggests a link between NP function, replication fidelity, and MuV pathogenesis, offering insights into viral replication and transcription processes.
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