JVI Accepts, published online ahead of print on 16 May 2007

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J. Virol. doi:10.1128/JVI.00580-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Surface exposed Adeno-associated virus Vp1-NLS capsid fusion protein rescues infectivity of non-infectious wild-type Vp2/Vp3 and Vp3-only capsids, but not 5-fold pore mutant virions

Joshua C. Grieger, Jarrod S. Johnson, Brittney Gurda-Whitaker, Mavis Agbandje-McKenna, and R. Jude Samulski^*

Curriculum in Genetics and Molecular Biology, Gene Therapy Center and Department of Pharmacology at University of North Carolina, Chapel Hill, North Carolina 27599, USA; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610

^* To whom correspondence should be addressed. Email: rjs{at}med.unc.edu.

Over the past couple of decades significant effort has been dedicated to the development of Adeno-associated virus (AAV) as a vector for human gene therapy. However, the 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-termini, this region plus the recently identified NLS (Grieger et. al. 2006 J. Virol 80:5199-210), and the virion pore at the 5-fold axis in infection. We generated two Vp1-fusion proteins (Vp1 and Vp1NLS) linked to the 8 kDa chemokine domain of rat fractokine (FKN) for the purpose of surface exposing upon assembly of the virion as previously described (Warrington et al. 2004 J. Virol 78:6595-609). The unique Vp1 N-termini were found to be exposed on the surface of these capsids and maintained their phospholipase A2 (PLA2) activity as determined by native dot blot westerns and PLA2 assays, respectively. Incorporation of the fusions into AAV2 capsids lacking a wtVp1, i.e. Vp2/Vp3 and Vp3-capsid only, increased infectivity by 3 to 5 fold (Vp1FKN) and 10-100 fold (Vp1NLSFKN), respectively. However, the surface exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, Leu336Trp) located at the base of the 5-fold 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 local affects within the pore as previously suggested.

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]