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Journal of Virology, August 2007, p. 7833-7843, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00580-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions{triangledown}

Joshua C. Grieger,1,2 Jarrod S. Johnson,2,3 Brittney Gurda-Whitaker,4 Mavis Agbandje-McKenna,4 and R. Jude Samulski1,2,3*

Curriculum in Genetics and Molecular Biology,1 Gene Therapy Center,2 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599,3 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 326104

Received 19 March 2007/ Accepted 8 May 2007

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.

* Corresponding author. Mailing address: Gene Therapy Center, University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, NC 27599-7352. Phone: (919) 962-1224. Fax: (919) 966-0907. E-mail: rjs{at}med.unc.edu

{triangledown} Published ahead of print on 16 May 2007.

Journal of Virology, August 2007, p. 7833-7843, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00580-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Johnson, J. S., Samulski, R. J. (2009). Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus. J. Virol. 83: 2632-2644 [Abstract] [Full Text]  
  • DiPrimio, N., Asokan, A., Govindasamy, L., Agbandje-McKenna, M., Samulski, R. J. (2008). Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly. J. Virol. 82: 5178-5189 [Abstract] [Full Text]  


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