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Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation
1Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029.
Insights
Conserved cysteine residues in influenza A virus hemagglutinin (HA) are crucial for infectious virus formation. Mutations show that cysteine at position 563 is essential, while others can attenuate the virus, suggesting a role for palmitylation.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Influenza A virus hemagglutinin (HA) possesses conserved cysteine residues at its carboxy terminus.
- These cysteine residues are known to be modified by palmitic acid (palmitylation) in H2, H3, and H7 HA subtypes.
Purpose of the Study:
- To investigate the role of acylated cysteines in the cytoplasmic tail of HA in the formation of infectious influenza A viruses.
- To determine the specific function of conserved cysteine residues in HA subtypes.
Main Methods:
- Reverse genetics techniques were employed to introduce mutations into the HA of influenza A/WSN/33 virus (H1 subtype).
- Site-directed mutagenesis was used to alter specific cysteine residues (C563, C560, C553) and combinations thereof.
- Phenotypic analysis of mutated viruses was performed in cell cultures to assess infectivity and attenuation.
Main Results:
- Mutation of cysteine at position 563 (C563) to other amino acids abolished infectious-particle formation.
- Mutations at C560 (to alanine or tyrosine) resulted in attenuated virus strains.
- Altering cysteine at position 553 (C553) to serine or alanine did not significantly impact viral phenotype.
- Double and triple cysteine mutations, eliminating palmitylation sites, also failed to produce infectious virus.
- Revertants of the attenuated C560 mutant consistently reverted to cysteine, not other amino acids.
Conclusions:
- The conserved cysteine residues in the HA cytoplasmic tail, particularly C563, play a critical role in the formation of infectious influenza A virus particles.
- Palmitylation of these cysteines is strongly suggested to be functionally important for viral infectivity.
- While palmitylation is indicated, structural constraints within the HA cytoplasmic tail cannot be definitively excluded as a factor in infectious particle formation.
Abstract:
The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.