Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Abstract

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.A deno-associated virus (AAV) is a nonenveloped singlestranded DNA virus that has a relatively simple capsid consisting of 3 proteins that share a C-terminal amino acid sequence (2). The three capsid proteins (VP1, VP2, and VP3) assemble in an approximate ratio of 1:1:10 to form an icosahedral particle ϳ246 nm in diameter. Recently the X-ray crystal structure of AAV serotype 8 (AAV8) identified a surface region that undergoes a conformational shift when the virus is incubated at acidic pH (14). This region of four amino acids (the pH quartet) is formed from three different symmetry-related capsids at the intersection of the 2-, 3-, and 5-fold axes. Two of these amino acids, E566 and Y707, interact through hydrogen bonds at pH 7.4 but progressively migrate from each other as the pH is lowered to 6.0 or 5.5, which are the approximate pHs of early and late endosomes. Similar amino acid rearrangements have been seen in the crystal structures of AAV1 (unpublished observation). In AAV2 (Fig. 1), E563 is structurally equivalent to E566 in AAV8, and it is flanked by two other acidic residues (E562 and E564) that are conserved in all AAV serotypes that have been sequenced to date (Table 1). In addition, a tyrosine (Y704), arginine (R389), and histidine (H526) are also highly conserved, as well as another acidic cluster (528DDEE531) near this region ( Fig. 1 and Table 1). Acidification of...

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

“…Our data suggest that at neutral pH, this region is not highly dynamic, raising the possibility that the role of autohydrolysis is to increase dynamics around the 5-fold axis during trafficking. Our results support the hypothesis that proteolysis is a trigger for externalization of VP1u, as was also recently suggested based on the identification of a protease function in AAV2 (39).…”

“…An additional factor that must be considered is autohydrolysis of AAV VPs. This was recently described for nine of the AAV serotypes (39). Bands originating from the degradation of VPs increase with time on SDS-PAGE gels.…”

“…It is believed that during cellular entry by AAV capsids, exposure to acidic conditions in endosomes leads to a conformational change and exposure of the VP1u and VP1/2 common regions required for endosomal escape and nuclear entry (34). Interaction with cellular proteases and pH-activated autohydrolysis have also been implicated in the entry process (39). In vitro studies typically use a heat pulse as a surrogate for cellular triggers of VP1u and VP1/2 externalization, although characterization of capsids after heating has primarily been limited to antibody detection and electron microscopy (EM) studies (34,(40)(41)(42)75).…”

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

“…It is important to note, however, that the previous study utilized AAV2 virions that had not gone through physiological endosomal processing, allowing for the conformational change that exposes the basic regions along with the PLA2 domain required for transduction. A recent study by Salganik et al showed that purified AAV2 virions have external protease activity at physiological pH that is ablated at low pH (76). It is possible that virions that traffic to the nucleus using alternative pathways can exert alternative functions, such as proteasemediated, NPC-independent nuclear entry under certain conditions.…”

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages

“…Small differences in the structures and dynamics of various parvovirus and adeno-associated virus (AAV) capsids have been reported (35,47,48). Although the crystal structures of empty particles or virus-like particles (VLPs) appear structurally highly similar to those of infectious DNA-containing capsids when different crystal structures are compared, some differences in the structures and dynamics have been revealed (49)(50)(51).…”