Identification of functionally important negatively charged residues in the @entity1473 end of @entity19 XXXX coronavirus A59 nucleocapsid protein.

@entity1522 @entity2574 @entity19 @entity535 @entity99 @entity493 @entity369 @entity1473
The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged @entity1522 are located within domain III in the @entity1473 end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein @entity1473 tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of @entity493 and other charged-residue substitutions were introduced in place of those in the N gene within a @entity19 @entity99 coronavirus A59 infectious clone. @entity2574 residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged @entity1522 . When either @entity535 was replaced by a positively charged residue or both were changed to @entity493 , viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to- @entity369 change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly.
