PMID- 19109385
OWN - NLM
STAT- MEDLINE
DCOM- 20090324
LR  - 20231213
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Print)
IS  - 0022-538X (Linking)
VI  - 83
IP  - 6
DP  - 2009 Mar
TI  - Enhancement of adeno-associated virus infection by mobilizing capsids into and 
      out of the nucleolus.
PG  - 2632-44
LID - 10.1128/JVI.02309-08 [doi]
AB  - Adeno-associated virus (AAV) serotypes are being tailored for numerous 
      therapeutic applications, but the parameters governing the subcellular fate of 
      even the most highly characterized serotype, AAV2, remain unclear. To understand 
      how cellular conditions control capsid trafficking, we have tracked the 
      subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal 
      immunofluorescence, three-dimensional infection analysis, and subcellular 
      fractionation. Here we report that a population of rAAV2 virions enters the 
      nucleus and accumulates in the nucleolus after infection, whereas empty capsids 
      are excluded from nuclear entry. Remarkably, after subcellular fractionation, 
      virions accumulating in nucleoli were found to retain infectivity in secondary 
      infections. Proteasome inhibitors known to enhance transduction were found to 
      potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases 
      transduction, mobilized virions into the nucleoplasm, suggesting that two 
      separate pathways influence vector delivery in the nucleus. Using a small 
      interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins 
      B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, 
      affect trafficking and transduction efficiency. Similar to effects observed with 
      proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated 
      nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to 
      effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the 
      nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both 
      pathways simultaneously using drug and siRNA combinations was synergistic and 
      increased transduction over 50-fold. Taken together, these results support the 
      hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in 
      the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic 
      sites likely permits uncoating and subsequent gene expression or genome 
      degradation. In summary, with these studies we have refined our understanding of 
      AAV2 trafficking dynamics and have identified cellular parameters that mobilize 
      virions in the nucleus and significantly influence AAV infection.
FAU - Johnson, Jarrod S
AU  - Johnson JS
AD  - Gene Therapy Center and Department of Pharmacology, The University of North 
      Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, North 
      Carolina 27599-7352, USA.
FAU - Samulski, R Jude
AU  - Samulski RJ
LA  - eng
GR  - F31 NS060688/NS/NINDS NIH HHS/United States
GR  - 1F31NS060688-01A1/NS/NINDS NIH HHS/United States
GR  - 5F31NS060688-02/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20081224
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (NPM1 protein, human)
RN  - 0 (Nuclear Proteins)
RN  - 0 (Phosphoproteins)
RN  - 0 (RNA-Binding Proteins)
RN  - 117896-08-9 (Nucleophosmin)
SB  - IM
MH  - Capsid/*metabolism
MH  - Cell Nucleolus/virology
MH  - Cell Nucleus/virology
MH  - Cytoplasm/chemistry
MH  - Dependovirus/genetics/*physiology
MH  - Gene Knockdown Techniques
MH  - HeLa Cells
MH  - Humans
MH  - Nuclear Proteins/antagonists & inhibitors
MH  - Nucleophosmin
MH  - Phosphoproteins/antagonists & inhibitors
MH  - RNA-Binding Proteins/antagonists & inhibitors
MH  - *Transduction, Genetic
MH  - Nucleolin
PMC - PMC2648275
EDAT- 2008/12/26 09:00
MHDA- 2009/03/25 09:00
PMCR- 2009/09/01
CRDT- 2008/12/26 09:00
PHST- 2008/12/26 09:00 [entrez]
PHST- 2008/12/26 09:00 [pubmed]
PHST- 2009/03/25 09:00 [medline]
PHST- 2009/09/01 00:00 [pmc-release]
AID - JVI.02309-08 [pii]
AID - 2309-08 [pii]
AID - 10.1128/JVI.02309-08 [doi]
PST - ppublish
SO  - J Virol. 2009 Mar;83(6):2632-44. doi: 10.1128/JVI.02309-08. Epub 2008 Dec 24.