Abstract
ABSTRACTWe previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus.IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of a nonenveloped viral capsid that appears to have a role in promoting transcription. A total of six mutants at the AAV capsid 2-fold interface were shown to have a severe defect in expressing their genomes, and the defect was at the level of mRNA accumulation. This suggests that AAV capsids have a novel role in promoting the transcription of the genomes that they have packaged. Since wt virions could not complement the mutant viruses, and the mutant viruses did not effectively inhibit wt gene expression, our results suggest that the capsid exerts its effect on transcription in cis.
Highlights
Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses
Data from our studies suggest that the 2-fold region of the associated virus (AAV) capsid has a role in transcription of the viral genome
Recent unpublished work from our laboratory identified K692A as the likely cause of the defect in mut47. This mutant had previously been characterized as being defective for capsid assembly but was subsequently found instead to be defective for binding the A20 antibody, which was the assay used for capsid assembly in the original study by Wu et al We focused our analysis on the six mutants D528A, D529A, K692A, E562A, E564A, and mut22 (Fig. 1)
Summary
Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A total of six mutants at the AAV capsid 2-fold interface were shown to have a severe defect in expressing their genomes, and the defect was at the level of mRNA accumulation This suggests that AAV capsids have a novel role in promoting the transcription of the genomes that they have packaged. We and others [3, 4] have found a number of mutants that are capable of assembling intact capsids but have a profound defect in infectivity (3 to Ͼ5 logs) These mutants cluster in a 30-Å-diameter region adjacent to the 2-fold interface on the capsid surface (Fig. 1) that has been called the “dead zone” [3]. Data from our studies suggest that the 2-fold region of the AAV capsid has a role in transcription of the viral genome
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