Surface-exposed Vp1-NLS fusion proteins increased infectivity of Vp2/Vp3 and Vp3-only AAV capsids by 10- to 100-fold, but failed to rescue infectivity in virions with fivefold pore Leu336 mutations.
Surface-exposed Vp1 fusion proteins can rescue infectivity of AAV capsids lacking wild-type Vp1, but not those with mutations at the fivefold pore, suggesting the pore plays a role in global structural changes essential for infectivity.
Estimación del efecto: 10- to 100-fold increase
Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.
Grieger et al. (Thu,) conducted a other in Adeno-associated virus (AAV) infectivity. Vp1-NLS Capsid Fusion Protein vs. Wild-type AAV2, Vp2/Vp3-only, Vp3-only, and Leu336 mutant virions was evaluated on Infectivity (luciferase transduction) (10- to 100-fold increase). Surface-exposed Vp1-NLS fusion proteins increased infectivity of Vp2/Vp3 and Vp3-only AAV capsids by 10- to 100-fold, but failed to rescue infectivity in virions with fivefold pore Leu336 mutations.