**4. Cell entry mechanism of AAV**

The major cell entry mechanism for AAV is via endocytosis utilizing clathrin-coated pits, although other minor mechanisms are possibly involved in this process. However, these alternative minor mechanisms are yet to be confirmed [6]. Upon AAV binding to its cell surface receptors, it stimulates intracellular signaling pathways, which in turn stimulates internalization of AAV. This phenomenon can be clearly explained using the mechanisms reported for AAV2 host cell interaction. It was shown that attachment of AAV2 to HSPG and αVβ5 integrin resulted in the activation of Rac1, an intracellular small guanosine triphosphate (GTP)-binding protein, and phosphoinositide 3-kinase (PI3K) in HeLa cells within 5 minutes of AAV2 infection [26]. Furthermore, inhibition of Notch1 by siRNA, a transmembrane receptor known to be involved in the activation of Rac1 and PI3K, was reported to decrease cell transduction by AAV2 [27], suggesting that the Rac1-PI3K pathway is necessary to initiate endocytosis of AAV2. Direct injection of AAV into the cytoplasm and nucleus of cells results in a significant lower infection rate than cells that are simply exposed to virus [28], suggesting that the processing of AAV virion through endosomal compartments is a critical initiating step for transduction following endocytosis.

In addition, transduction efficiency of AAV is largely dependent on the endosomal pH. Changing the pH to acidic (pH 4–6) inside the endosomal compartment facilitates transduction of AAV, whereas blocking acidification during endosomal processing decreases the rate of transduction [29–31]. Also, the application of different classes of proteasome inhibitors such as tripeptidyl aldehydes and N-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL) and the anthracycline compounds such as doxorubicin increases the rate of viral translocation to the nucleus [32]. Furthermore, LLnL appears to increase AAV2 capsid ubiquitination that results in augmented gene transfer in different cell types [33], suggesting a mechanism by which these inhibitors increase transduction may be related to ubiquitination. AAV must exit from the endosome first before translocating to the nucleus. Prior to escape from the endosome, AAV undergoes a conformational change leading to the exposure of the unique N-terminal ends of VP1 and VP2, which contains a domain of phospholipase A2 (PLA2) [34], an enzyme that breaches the endosomal membrane and thereby facilitates efficient endosomal escape of viral particles. Upon endosomal escape, AAV enters the nucleus as an intact particle [28] and uncoating then occurs inside the nucleus. However, nuclear transport of AAV is a slow process, approximately only 1–2% of internalized AAV enters and expresses in the nucleus, and the whole entry process takes about 2–13 h [35]. Thus, most viral particles which fail to translocate are located outside or away from the nucleus.

The viral particles that fail to translocate into the nucleus are eventually degraded by host proteasomes in the cytoplasm and presented as antigen to cytotoxic T cells via the major histocompatibility complex (MHC) class I pathway [36]. Although there are several studies that have investigated the nuclear entry of AAV, the mechanism by which AAV translocates into the nucleus is still unclear. Because AAV is a small virus with a diameter of around 20 nm, it has been suggested that the virion enters the nucleus using the nuclear pore complex (NPC) [37].

Furthermore, the nuclear entry of AAV is dependent on importin-β, a nuclear import protein that has been shown to play a key role in facilitating the binding of viral particles to host nuclei in other viral infectious pathways [38, 39]. Another study using single-point edge excitation sub-diffraction (SPEED) microscopy, a form of super-resolution imaging, to track single AAV particles revealed that approximately 17% of AAV particles were imported through the NPC successfully to the nucleus [40], reinforcing the importance of the NPC in AAV nuclear transfer. Interestingly, there is further evidence that nucleolin, a protein that shuttles between cytoplasm and nucleus, specifically binds to AAV capsid, which suggests that nucleolin may act as a nuclear receptor for AAV particles as well [41]. Upon entry into the nucleus, the ssDNA of AAV genome is converted to double-stranded DNA (dsDNA) using nuclear machinery of the target cells for transcription of the transgene [42]. The synthesis of second DNA strand has been considered as a rate-limiting factor for the onset and efficiency of transgene expression in ssAAV vectors [43]. As a result, second-generation AAV vectors with a dsDNA, also known as self-complementary AAV (scAAV) vectors, have been developed to improve the transduction and transcription efficiency. In the past decade, several studies have shown that new scAAV vectors provide safe, reliable, and organ-specific transduction both *in vitro* [44–46] and *in vivo* [46–49]. This suggests that the limitations associated with cell transduction using ssAAV genome can be overcome by the use of scAAV vectors in gene therapy.
