Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus

Abstract

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

The interferon-ɣ inducible factor 16 (IFI16) restricts adeno-associated virus serotype 2 (AAV2) transduction in an immune-modulatory independent way

“…S6 in the supplemental material). However, the image-based quantification of the post-transcriptional silencing of IFI16 revealed that neither partial uncoating in the cytoplasm, complete uncoating in the nucleolus, or cell cycle progression (32) was affected (data not shown).…”

“…6, B and D). The DAPI based cell cycle analysis workflow was adapted from the protocol published by Roukos et al, 2015 (31) and validated as described previously (32). Both assays revealed an increase in the number of cells in G2 cell cycle phase upon AAV2 (Fig.…”

Non-canonical induction of autophagy increases adeno-associated virus type 2 (AAV2) transduction efficiency

“…The absence of co-release of larger molecules during parvovirus endosomal escape indicates that the virus-induced membrane rupture is limited and does not involve complete lysis (43). Specific to AAV2, it was demonstrated that the pH-dependent conformational changes of the AAV2 capsid results in the exposure of PLA 2 and is important for AAV2 infection, partial uncoating (45), endosomal escape (14), and nuclear entry in particular (46). However, inflicting endocytic membrane damage results either in a galectin-mediated membrane damage response pathway or in the induction of autophagy.…”

Liver directed adeno‐associated viral vectors to treat metabolic disease

“…29 Inside the nucleus, the AAV capsid uncoats releasing the single-stranded DNA (ssDNA) genome. 30 The ssDNA genome is converted to a double-stranded intermediate by the host cell's DNA repair machinery, which then circularizes via the ITRs. 31 These circular AAV genomes are known as "episomes," a stable non-integrating form of the viral cargo that can exist as circular monomers or concatamers.…”

“…Next, basic clusters in VP1 and VP2 subunits act as nuclear localization sequences to direct the AAV capsid into the nucleus 29 . Inside the nucleus, the AAV capsid uncoats releasing the single‐stranded DNA (ssDNA) genome 30 . The ssDNA genome is converted to a double‐stranded intermediate by the host cell's DNA repair machinery, which then circularizes via the ITRs 31 .…”