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A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Importance of the penultimate positive charge in mouse hepatitis coronavirus A59 membrane protein
Sandhya Verma1, Lisa A Lopez, Valerie Bednar
1Biodesign Institute, Center for Infectious Diseases and Vaccinology, PO Box 875401, Arizona State University, Tempe, AZ 85287-5401, USA.
Abstract:
The coronavirus membrane (M) protein carboxy tail interacts with the nucleocapsid during virus assembly. Previous studies demonstrated that the two terminal residues are important, and the charged residue (R227) in the penultimate position in the mouse hepatitis coronavirus (MHV) A59 M protein was suggested to participate in intermolecular interactions with negative charges in the nucleocapsid (N) protein. To determine the significance of the positive charge at position 227, we substituted the arginine with lysine (K), aspartic acid (D), glutamic acid (E), or alanine (A) and studied these by reverse genetics in the context of a MHV full-length infectious clone. Viruses with wild-type phenotype were readily recovered with the K or A substitutions. In contrast, negative-charge substitutions were not tolerated as well. In all recovered R227D viruses the negative charge was replaced with heterologous residues resulting from apparent template switching during negative-strand synthesis of subgenomic RNA 7. An additional second-site compensatory V202I substitution was present in some viruses. Recovered R227E viruses had second-site changes within the M protein carboxy tail that were partially compensatory. Significantly, most of the second site changes in the R227E mutant viruses were previously shown to compensate for the removal of negative charges in the N protein. Our results strongly indicate that a positive charge is not absolutely required. It is clear that other regions within the tail must also be involved in helping mediate interactions between the M protein and the nucleocapsid.
Insights
A positive charge on the mouse hepatitis coronavirus (MHV) M protein is not essential for virus assembly. Other regions of the M protein tail also mediate interactions with the nucleocapsid.
Area of Science:
- Virology
- Molecular Biology
- Protein Interactions
Background:
- The coronavirus membrane (M) protein's carboxy tail is crucial for virus assembly, interacting with the nucleocapsid.
- Previous research highlighted the importance of the M protein's terminal residues, particularly a charged residue at position 227 in MHV-A59.
Purpose of the Study:
- To investigate the significance of the positive charge at position 227 of the MHV M protein for nucleocapsid interaction.
- To determine if substituting this charged residue affects virus assembly and function.
Main Methods:
- Utilized reverse genetics with a full-length MHV infectious clone.
- Introduced substitutions at position 227 of the M protein (Arginine to Lysine, Aspartic Acid, Glutamic Acid, or Alanine).
- Analyzed recovered viruses for phenotypic changes and identified second-site mutations.
Main Results:
- Viruses with Lysine or Alanine substitutions at position 227 were viable with wild-type phenotypes.
- Negative-charge substitutions (Aspartic Acid, Glutamic Acid) were poorly tolerated, leading to template switching or compensatory mutations.
- Second-site mutations in R227E viruses partially compensated for the loss of the positive charge and were previously shown to compensate for changes in the nucleocapsid protein.
Conclusions:
- A positive charge at position 227 of the MHV M protein is not strictly required for virus assembly.
- The carboxy tail of the M protein contains other regions involved in mediating interactions with the nucleocapsid.
- The findings suggest a complex interplay of residues within the M protein tail for effective nucleocapsid binding.
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