Since NP1 directly interact with RPA in vitro we set out to study whether NP1 colocalizes with RPA in cells First HEK293 cells transiently expressing Myc-t-agged NP1 proteins were used to examine the

Since NP1 directly interacts with RPA in vitro, we conducted a study to determine whether NP1 colocalizes with RPA in cells. We utilized HEK293 cells transiently expressing Myc-tagged NP1 proteins to examine the subcellular localization of NP1 and endogenous RPA by confocal microscopy. Our findings revealed that both NP1 and RPA were distributed throughout the nucleus and exhibited clear colocalization (Fig. 7A). Next, we monitored the colocalization of NP1 and RPA in HEK293 cells during the productive replication of AAV2. The results indicated that NP1 largely colocalizes with RPA at discrete sites within the nucleus (Fig. 7B), suggesting that viral replication could alter the distribution pattern of NP1 and RPA in the nucleus.

Considering the fact that NP1 is localized in the AAV2 replication center, we also examined the colocalization of RPA with the viral replication centers. As Rep 78 protein has been reported to colocalize with the AAV2 replication center, we immunostained for this protein to localize the virus replication centers. In plasmids containing the AAV2 duplex-genome, we inserted a FLAG-tag at the 5'-end of the Rep 78 coding sequence and used an anti-FLAG antibody to detect the localization of Rep 78 in the cells. Similar to NP1, we observed a precise form of colocalization between the anti-RPA70 staining and the anti-FLAG antibody staining in AAV2 dsDNA-transfected cells (Fig. 7C), indicating that RPA localizes within the virus replication centers.

Taken together, our study confirmed that NP1 and RPA not only associate together in vitro to form a stable complex, but also have a clear colocalization in vivo. Moreover, both proteins are recruited into the viral replication center during AAV2 productive replication