Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Abstract

Recombinant adeno-associated viral (rAAV) vectors have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggested that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains illdefined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently labeled particles revealed that rAAV2 localized to importin-␤-dense regions of cells in late trafficking steps. Additionally, small interfering RNA (siRNA) knockdown of importin-␤ partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50 to 70%. Furthermore, coimmunopreciptation (co-IP) analysis revealed that capsid proteins from rAAV2 could interact with importin-␤ and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with importin-␤. We tested several other serotypes and found that the extent of importin-␤ interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins, and the results suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with importin-␤ alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have an enhanced nuclear entry capability and transduction potential. IMPORTANCEUse of recombinant adeno-associated viral (rAAV) vectors for gene therapy applications is limited by relatively low transduction efficiency, in part due to cellular barriers that hinder successful subcellular trafficking to the nucleus, where uncoating and subsequent gene expression occur. Nuclear translocation of rAAV has been regarded as a limiting step for successful transduction but it remains ill-defined. We explored potential nuclear entry mechanisms for rAAV2 and found that rAAV2 can utilize the classical nuclear import pathway, involving the nuclear pore complex, the small GTPase Ran, and cellular karyopherins. These results could lend insight into the rational design of novel rAAV vectors that can more efficiently translocate to the nucleus, which may lead to more efficient transduction.

Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

“…The values in all panels represent mean and SD from triplicate experiments. (13)(14)(15). The identity of the subcellular compartment from which AAV escapes into the cytoplasm has not been established but may be of great importance for rational vector design because escape from the endosomal system might be a rate-limiting step in AAV transduction.…”

“…After internalization, the low pH in endosomes (8) and, possibly, the action of endosomal proteases (9) trigger a conformational change in the AAV capsid, exposing the N-terminal domain of the largest capsid protein, VP1, on the capsid surface (10). This so-called VP1 unique region (VP1 u ) harbors a phospholipase A2 (PLA2) domain and a bipartite nuclear localization signal, which are sequentially required for escape into the cytoplasm and the nuclear import of intact capsids (11)(12)(13)(14)(15). Both the escape into the cytoplasm and the nuclear import have been proposed to be rate limiting because only a small fraction of virions successfully reaches the nucleus, and the majority can instead be observed in a perinuclear vesicular compartment for extended periods (16)(17)(18).…”

Recombinant Adeno-Associated Virus Utilizes Cell-Specific Infectious Entry Mechanisms

“…Virus was produced in HEK293 cells as previously described (29,30). Briefly, polyethylenimine "max" (PEI) was used for the triple transfection of the pXR2/pXR2-R585E/pXR8 cap and rep plasmids, the pXX6-80 helper plasmid, and a terminal repeat (TR)-luciferase (Luc) reporter plasmid containing the firefly luciferase transgene flanked by inverted terminal repeats under the control of the chicken beta actin (CBA) promoter.…”

“…After identification of peak fractions by quantitative PCR (qPCR), virus was dialyzed into phosphate-buffered saline (PBS). Titers were calculated by qPCR according to established procedures (29,30) using a LightCycler 480 instrument and SV40pA primers previously utilized (31). For Cy5-labeled virus, purified rAAV2 was labeled with Cy5 dye (GE Amersham), extensively dialyzed, and examined for purity as previously described (29,32).…”

“…Titers were calculated by qPCR according to established procedures (29,30) using a LightCycler 480 instrument and SV40pA primers previously utilized (31). For Cy5-labeled virus, purified rAAV2 was labeled with Cy5 dye (GE Amersham), extensively dialyzed, and examined for purity as previously described (29,32). Single-stranded and self-complementary rAAV2-CMV-GFP (ssrAAV2-CMV-GFP and scrAAV2-CMV-GFP, respectively) were prepared and verified for purity as described previously (30).…”