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Analysis of a potential myristoylation site in hepatitis A virus capsid protein VP4
M Tesar1, X Y Jia, D F Summers
1Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Abstract:
The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.
Insights
Hepatitis A virus (HAV) VP4 protein myristoylation is not essential for viral replication. Studies show leader cleavage and myristoylation are not required for infectious HAV growth in cell cultures.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Myristoylation of the VP4 capsid protein is a known post-translational modification in some picornaviruses.
- This modification occurs at an N-terminal glycine residue after leader peptide cleavage, exposing a myristoylation signal.
- Hepatitis A virus (HAV) VP4 sequence analysis suggested a potential myristoylation site.
Purpose of the Study:
- To investigate the significance of the potential myristoylation signal in the Hepatitis A virus (HAV) VP4 protein.
- To determine if leader cleavage and VP4 myristoylation are necessary for HAV replication.
Main Methods:
- Engineered mutations in the HAV VP4 sequence to alter the consensus myristoylation motif and potential leader cleavage site.
- Constructed a mutant lacking the proposed leader sequence.
- Expressed wild-type and mutant HAV sequences in BS-C-1 cells and assessed leader cleavage, translation, and processing efficiency.
- Transfected cells with viral cDNA transcripts and evaluated infectious virus production and myristate incorporation.
Main Results:
- Leader cleavage was not observed in wild-type or mutant HAV proteins.
- A threonine to asparagine mutation reduced translation and processing efficiency.
- Infectious HAV was produced from both wild-type and mutagenized constructs.
- No detectable myristate incorporation was observed in any of the tested HAV variants.
Conclusions:
- Hepatitis A virus (HAV) does not require leader cleavage for VP4 protein.
- Myristoylation of VP4 is not essential for HAV growth and replication in cultured cells.
- These findings differentiate HAV replication mechanisms from other myristoylated picornaviruses.