Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

Abstract

A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>10 7 -fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3-to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome.A deno-associated virus (AAV) is a small parvovirus of the Dependovirus genus that is currently being tested as a gene therapy vector. Although it has been studied extensively, many questions remain about some of the basic processes governing AAV infection. One such process is the role of acidification of the endosomal compartments in AAV trafficking and uncoating. Several groups have shown that blocking endosomal acidification inhibits AAV2 infection and recombinant AAV (rAAV) transduction (1-4). Furthermore, Sonntag et al. (4) have demonstrated the need for AAV to traffic through the endosomal system, since microinjection of virus into the cytoplasm fails to lead to a productive infection. One hypothesis is that trafficking through the progressively acidic endosomes leads to the externalization of the VP1 and VP2 N termini, which contain both nuclear localization signal (NLS) domains and a phospholipase A2 (PLA2) domain that have been shown to be critical for infection (4-7). To this end, there is evidence that epitopes associated with the viral protein 1 (VP1)/ VP2 N termini become exposed early in infection, during a time that the vi...

Structure of neurotropic adeno-associated virus AAVrh.8

“…Interestingly, AAV8 Y707 and adjacent residues are involved in pH-mediated structural transitions predicted to occur during virus trafficking through the endocytic pathway (Nam et al, 2011), and E563A and H526A mutations in AAV2 (equivalent to E564 and H527 in AAVrh.8) showed severe defect in infectivity (Lochrie et al, 2006; Wu et al, 2000). Other studies with AAV2 have shown that an E563A mutation eliminates a novel capsid protease activity (Salganik et al, 2012), and Y704A (equivalent to AAVrh.8 Y705) plays a role in second-strand synthesis and transcription of the input genome (Salganik et al, 2014). AAV2 Y704 is also part of a series of tyrosines which when mutated to phenylalanines resulted in improved intracellular trafficking to the nucleus and hence increase in transduction efficiency dependent on the cell line (Zhong et al, 2008).…”

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

“…Although the exact capsid function being abrogated by A20 is unknown, overlap of its footprint with a transduction "dead zone" described for AAV2 and the negative effect of mutations of its epitope residues on tissue transduction efficiency suggest a block of an essential interaction(s) (38,(71)(72)(73). While the AAV1 infection step neutralized by ADK1b remains to be elucidated, its contact site includes residues 263, 265, 709, and 717, which have been reported to affect muscle tropism and transduction efficiency (71).…”

Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy

“…Additionally, the use of distinct capsids to pseudotype AAV vectors (48), such as serotypes 8 and 9, confers distinct patterns of tissue trophism in wild-type and disease states (49). Other capsid-specific properties may influence genotoxicity, such as subcellular localization (50), uncoating kinetics (51), and perhaps even transcription of the input genome (52). Preclinical testing should therefore be designed to account for the differential behavior of each vector-capsid uniquely and incorporate long-term observation for malignancies as well as genomic analyses to characterize vector integrations.…”