Samulski et al. replies—Using the procedure that Mizukami et al. used8, we also observed a 150-kDa protein (Fig. 1, lane 1); however, this method (described by Chong and Rose16) does not stringently purify plasma membrane proteins. In our paper2, we used a method that specifically enriched cell surface proteins by 30-fold (ref. 17), as assessed by 5'-nucleotidase activity. In these more stringent conditions, binding to the 150-kDa protein was not detected. (Fig. 1, lane 2), thus our submitted gel2 was truncated to save space. This protein may be a non-plasma membrane protein (for example, nucleolin as identified by Qiu and Brown), or a cell surface protein that migrates in a different fraction with our procedure. As the 'fold' enrichment of plasma membrane proteins was not monitored in Mizukami's study8, all interpretations are plausible.
Lanes 1 & 2 represent an AAV–2 overlay on equivalent amounts of membrane purified by the Chong & Rose method and the Hennache & Boulanger method respectively. Arrows point to the 150 kDa (lane 1) and 100 kDa (lane 2) proteins that interact with AAV.
As for β5 integrin, we also did not see interaction with the purified form, possibly because of the absence of essential post-translational modification. It should be noted that we observed AAV binding to immunoprecipated β5 integrin, supporting the specificity of this interaction. Furthermore, we established that there is a role for integrin in AAV-2 infection (ref. 2, Figs. 2 and 3). The presence of integrin influences viral infection, but is not essential, as is the case with adenovirus10,18. Figure 3 of our study2 clearly demonstrates that expression of β5 substantially increases AAV-2 internalization in a time-dependent manner, indicating a role in AAV entry, which may have important consequences in vivo2,10,18.
As for the transduction data, the 260% enhancement we observed is very similar to that seen for adenovirus (320%), whose use of αVβ5 integrin as a co-receptor is well established. In addition, it is not surprising that AAV may interact with integrin in a non-RGD manner. A ligand does not have to use an RGD or RGD-like motif in order to interact with integrin.
Integrin αVβ3 and αVβ5 facilitate adenovirus infection; however, it is αVβ5 integrin that has been shown to have a dual role in facilitating both membrane permeabilization and internalization17. In addition, compared with αVβ3 integrin, αVβ5 internalizes adenovirus at a faster rate and renders cells significantly more susceptible to infection18. These studies and our data strongly suggest that both Ad and AAV use αVβ5 as a co-receptor to mediate viral entry.
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Samulski, R., Bartlett, J. & Summerford, C. Adeno-associated virus 2 co-receptors?-second reply. Nat Med 5, 468 (1999). https://doi.org/10.1038/8330
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DOI: https://doi.org/10.1038/8330
