Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

Abstract

We 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.

The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction

“…Thus, the inability of AAV particles to traffic through the Golgi network prior to nuclear entry appears to correlate with a defect in genome transcription. These observations are corroborated by earlier reports that the AAV capsid is involved in regulating vector genome transcription ( 23 – 26 ).…”

Capsid Proteins are Necessary for Replication of a Parvovirus

“…Regardless of the actual mechanism, the decrease in VP protein production due to premature transcript termination causes a dramatic and unexpected shutdown of viral replication. We found no evidence of strong trans expression feedbacks between VP and NS proteins, as has been documented for other parvoviruses (30)(31)(32)(33)(34)(35)(36). In particular, the impact of decreased abundance in vp transcript and VP proteins on replication does not appear to involve a direct effect on NS1 or NS2.…”

Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction

“…A role for residues in the 2/5-fold wall in receptor attachment has been described for CPV [ 58 , 59 , 60 ]. Importantly, a role for 2-fold residues in altering capsid trafficking and transcription has been described for dependoparvoviruses [ 72 , 73 ]. Thus, the hypothesis is that the 2-fold and its surrounding residues control rodent protoparvovirus tumor tropism, as well as cell killing efficiency.…”