title: Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.
abstract: The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.
authors.affiliation: Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Nagesh
authors.initials: N
authors.lastname: Pulicherla
authors.forename: Pradeep
authors.initials: P
authors.lastname: Kota
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D000229:Dependovirus / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014771:Virion / Q000737:chemistry / Q000378:metabolism
chemical_list: D036022:Capsid Proteins