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Reovirus nonstructural protein muNS binds to core particles but does not inhibit their transcription and capping
T J Broering1, A M McCutcheon, V E Centonze
1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Abstract:
Previous studies provided evidence that nonstructural protein muNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455-466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of muNS. Given the possible role of muNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant muNS can bind to cores in vitro. The muNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of muNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of muNS were bound. Thus, the muNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the muNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to muNS. The muNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing muNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins mu1 and sigma3. These findings are consistent with the presence of muNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, muNS may block or delay outer-capsid assembly and allow continued transcription by these particles.
Insights
Mammalian reovirus nonstructural protein muNS binds to viral cores, potentially delaying outer capsid assembly. This interaction allows transcriptase particles to continue synthesizing viral RNA within infected cells.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Nonstructural protein muNS is found in mammalian reovirus assembly intermediates.
- These intermediates contain core-like particles and muNS, and can synthesize viral RNA transcripts.
- The role of muNS in assembly and transcription is suggested but not fully understood.
Purpose of the Study:
- To investigate the in vitro interaction between recombinant muNS and viral cores.
- To determine the effect of muNS binding on core structure and function.
- To assess the potential role of muNS in regulating viral particle assembly and transcription.
Main Methods:
- In vitro binding assays using recombinant muNS and purified viral cores.
- Buoyant density analysis of muNS-core complexes.
- Negative-stain electron microscopy to visualize muNS-core complexes.
- In vitro transcription assays to assess the activity of muNS-bound cores.
- In vitro competition assays with outer-capsid proteins.
Main Results:
- Recombinant muNS binds to viral cores but not to virions or intermediate subvirion particles.
- muNS binding to cores does not show saturation or defined stoichiometry, reducing particle buoyant density.
- Electron microscopy reveals intact cores linked by amorphous muNS density.
- muNS-bound cores retain RNA transcriptase and 5' capping activity.
- muNS binding to cores inhibits the stoichiometric binding of outer-capsid proteins mu1 and sigma3.
Conclusions:
- The muNS protein interacts with reovirus cores, forming complexes that retain transcriptase activity.
- muNS binding to cores may prevent or delay the assembly of outer capsid proteins.
- This mechanism allows transcriptase particles to continue RNA synthesis within infected cells.