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The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

ABSTRACT: Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo.

SUBMITTER: Holscher C

PROVIDER: S-EPMC4666624 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

Hölscher Christina C Sonntag Florian F Henrich Katharina K Chen Qingxin Q Beneke Jürgen J Matula Petr P Rohr Karl K Kaderali Lars L Beil Nina N Erfle Holger H Kleinschmidt Jürgen A JA Müller Martin M

PLoS pathogens 20151201 12

Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including ...[more]

PMID: 26625259

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Project description:Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

Project description:Central nervous system (CNS) transduction by systemically administered recombinant adeno-associated viral (AAV) vectors requires crossing the blood-brain barrier (BBB). We recently mapped a structural footprint on the AAVrh.10 capsid, which, when grafted onto the AAV1 capsid (AAV1RX), enables viral transport across the BBB; however, the underlying mechanisms remain unknown. Here, we establish through structural modeling that this footprint overlaps in part the sialic acid (SIA) footprint on AAV1. We hypothesized that altered SIA-capsid interactions may influence the ability of AAV1RX to transduce the CNS. Using AAV1 variants with altered SIA footprints, we map functional attributes of these capsids to their relative SIA dependence. Specifically, capsids with ablated SIA binding can penetrate and transduce the CNS with low to moderate efficiency. In contrast, AAV1 shows strong SIA dependency and does not transduce the CNS after systemic administration and, instead, transduces the vasculature and the liver. The AAV1RX variant, which shows an intermediate SIA binding phenotype, effectively enters the brain parenchyma and transduces neurons at levels comparable to the level of AAVrh.10. In corollary, the reciprocal swap of the AAV1RX footprint onto AAVrh.10 (AAVRX1) attenuated CNS transduction relative to that of AAVrh.10. We conclude that the composition of residues within the capsid variable region 1 (VR1) of AAV1 and AAVrh.10 profoundly influences tropism, with altered SIA interactions playing a partial role in this phenotype. Further, we postulate a Goldilocks model, wherein optimal glycan interactions can influence the CNS transduction profile of AAV capsids.IMPORTANCE Understanding how viruses cross the blood-brain barrier can provide insight into new approaches to block infection by pathogens or the ability to exploit these pathways for designing new recombinant viral vectors for gene therapy. In this regard, modulation of virus-carbohydrate interactions by mutating the virion shell can influence the ability of recombinant viruses to cross the vascular barrier, enter the brain, and enable efficient gene transfer to neurons.

Dataset Information

The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

Publications

The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

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