Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

Abstract

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

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

“…In contrast, protease activity on an external substrate was seen only at pH 7.5, not at pH 5.5. Moreover, external protease activity was completely eliminated by the E563A mutation in AAV2, an amino acid structurally equivalent to one that had previously been seen to undergo a structural change when AAV8 crystals were examined at pH 5.5 (14). In contrast, autolytic cleavage appeared not to be affected by this mutation.…”

“…These amino acids are potentially interacting through hydrogen bonding. In AAV8, the equivalent amino acids to Y704 and E563 change their positions at acidic pH (14). Also present are two highly conserved glutamates (E562 and E563) and a second charged cluster that contributes E531, all in stick form.…”

“…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.…”

Clathrin-Associated Endocytosis as a Route of Entry into Cells for Parvoviruses

“…This destabilization may lead to activation of the PLA2 activity for endosomal escape and the NLS for nuclear targeting. As noted, it was observed by Nam et al (2011) that at pH 4.0 specific interactions between the capsid and the packaged DNA genome are weakened but it is insufficient for genome uncoating. Rather, the intracapsid genome may be compacted.…”

“…AAV mutants that are consistent with a nuclear localization signal-deficient phenotype, traffick through the early endosome as the recombinant AAV2 control does, but forms a more diffuse accumulation pattern in the perinuclear area consistent with an NLS defect . In a recent study, AAV-8 was exposed to pHs ranging from 7.5 to 4.0 and crystal structures of empty particles and green fluorescent protein gene-packaged particles were analyzed (Nam et al, 2011). The capsid surface topologies of particles exposed to various pHs were similar except changes located close to the two-fold depression and significant amino acid side chain conformational changes were seen on the interior surface of the capsid under the three-fold axis.…”