Endocytosis and Nuclear Trafficking of Adeno-Associated Virus Type 2 Are Controlled by Rac1 and Phosphatidylinositol-3 Kinase Activation

Abstract

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that causes no currently known pathology in humans. Despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. In this study, we have examined endocytic and intracellular trafficking of AAV-2 using fluorescent (Cy3)-conjugated viral particles and molecular techniques. Our results demonstrate that internalization of heparan sulfate proteoglycan-bound AAV-2 requires ␣V␤5 integrin and activation of the small GTP-binding protein Rac1. Following endocytosis, activation of a phosphatidylinositol-3 (PI3) kinase pathway was necessary to initiate intracellular movement of AAV-2 to the nucleus via both microfilaments and microtubules. Inhibition of Rac1 using a dominant N17Rac1 mutant led to a decrease in AAV-2-mediated PI3 kinase activation, indicating that Rac1 may act proximal to PI3 kinase during AAV-2 infection. In summary, our results indicate that ␣V␤5 integrinmediated endocytosis of AAV-2 occurs through a Rac1 and PI3 kinase activation cascade, which directs viral movement along the cytoskeletal network to the nucleus.

Intracellular trafficking of adeno-associated viral vectors

“…39 To this end, soluble heparin can compete for binding of AAV2 to the surface of many cell types. 39,40 Mutagenesis and crystal structure studies have identified the amino acids in the capsid of AAV2 responsible for HSPG binding. 41,42 Heparin has also been reported to compete for AAV3 binding to cells, implying that AAV2 and AAV3 may share HSPG as an attachment receptor.…”

“…52,53 Nonetheless, inhibition of aVb5 integrin with blocking antibodies can prevent internalization of bound rAAV2 on the surface of HeLa cells. 40 Similarly, platelet-derived growth factor receptor (PDGFR) has been considered the coreceptor for AAV5 and is also required for efficient infection with this serotype. 54 However, PDGFR itself is a sialo-glycoprotein containing both N-and O-linked oligosaccharide chains with sialic acid.…”

“…For example, rAAV2 binding to HSPG and aVb5 integrin stimulates activation of Rac1, an intracellular small GTP-binding protein. 40 The activation of Rac1 appears to be necessary to initiate endocytosis of cell-surface-bound AAV2, since expression of the dominant-negative N17Rac1 mutant inhibits endocytosis of virus. 40 In this context, inhibition of either Rac1 or aVb5 integrin prevents endocytosis of AAV2, implying a potential functional link between these two signaling pathways in AAV2 infection.…”

“…40 The activation of Rac1 appears to be necessary to initiate endocytosis of cell-surface-bound AAV2, since expression of the dominant-negative N17Rac1 mutant inhibits endocytosis of virus. 40 In this context, inhibition of either Rac1 or aVb5 integrin prevents endocytosis of AAV2, implying a potential functional link between these two signaling pathways in AAV2 infection. The activation of Rac1 appears to be closely linked to subsequent activation of the phosphatidylinositol-3 kinase pathway that is required for movement of AAV particles to the nucleus along microtubules and/or microfilaments.…”

“…The activation of Rac1 appears to be closely linked to subsequent activation of the phosphatidylinositol-3 kinase pathway that is required for movement of AAV particles to the nucleus along microtubules and/or microfilaments. 40 Endosomal processing and trafficking of AAV It has become increasingly recognized that following endocytosis, processing of rAAV virions through the endosomal compartment is intricately linked to transduction. There are several components of endosomal processing that likely determine the efficiency of transduction with a given serotype and receptor-entry pathway.…”