Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.
Bibliographical noteFunding Information:
We thank members of the Carette, Qiu, and Chapman laboratories for discussions and support and Juliana Idoyaga (Stanford University) for flow cytometer access and discussions. S.P., W.Z., M.S.C., J.Q., K.E.B., J.F.E., and J.E.C. were responsible for the overall design of the study. S.P. designed and constructed swap mutants and performed all genetic complementation and mutant studies. J.E.C. designed deletion and glycosylation mutants, J.E.W. constructed glycosylation mutants, A.S.P. performed mutant immunoblots, and O.D. contributed to the production of E. coli ectodomain constructs. W.Z. and J.Q. prepared membrane proteins and purified AAV-BP. W.Z. prepared purified AAV stocks, and J.Q. and F.C. constructed PKD-expressing plasmids in E. coli and performed all virus overlay assays. S.S.G. purified His-tagged PKD proteins. X.D. and Z.Y. performed virus neutralization luciferase assays, and N.L.M. performed the fluorescence neutralization assay. S.P., J.E.C., and J.Q. wrote the manuscript, with intellectual input from M.S.C., J.F.E., and Z.Y., and W.Z. revised the manuscript. The work was funded in part by NIH grants R01 GM066875 (M.S.C.), R35 GM122564 (M.S.C.), DP2 AI104557 (J.E.C.), U19 AI109662 (J.E.C.), R01 AI070723 (J.Q.), R21 AI112803 (J.Q.), and P01 HL051670 (J.F.E.); Cystic Fibrosis Foundation grant YAN15XX0
(Z.Y.); the Weston Havens Foundation (J.E.C., S.P., and A.S.P.); and the Stanford SPARK program (S.P. and A.S.P.). J.E.C. is a David and Lucile Packard Foundation fellow. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
© 2017 American Society for Microbiology.
- Adeno-associated virus
- Gene therapy
- Receptor-ligand interaction
- Viral receptor
- Virus overlay assay
- Virus-host interactions