The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses

<div><p>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 <i>in vitro</i> and <i>in vivo</i> 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 <i>in vitro</i> and <i>in vivo</i>.</p></div

Knockdown of enzymes of the SUMOylation pathway does not alter expression of a stably integrated CMV-eGFP gene.

<p>A HeLa cell line stably expressing eGFP was transfected with four different siRNAs targeting Ubc9 and Sae2, respectively. Forty-eight h later, the cells were infected with scAAV2-renilla luciferase at an MOI_GC of 10³. Twenty-four h after infection, cells were harvested by trypsinization. One half of a well of a 6-well plate was proceeded for FACS-analysis (a), the other half was used to determine luciferase activity (b). Mean values and standard deviation of two independent experiments of mean fluorescent intensity (MFI) and relative light units (RLU), respectively, are shown. The values obtained after transfection with <i>AllStars negative control siRNA</i> (Qiagen; ‘scrambled’) were set to 100 in both cases.</p

Prediction of putative SUMOylation sites in the AAV VP1 proteins.

<p>Potential SUMOylation sites and SUMO-interacting motifs (SIM) were predicted using GPS-SUMO [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005281#ppat.1005281.ref077" target="_blank">77</a>]. Number and specific position of potential SUMOylation sites and SIMs are shown on the left hand side. AAV1, 2, 3, 6, 7, 8, 9, 10 and 13 harbor a potential lysine (K) which can serve as SUMOylation target (yellow). Also AAV4, 11 and 12 expose a potential SUMOylation target K at a position nearby (green).</p

Knockdown of SUMOylation key enzymes increased transduction with different serotypes and capsid variants but not that of autonomous parvovirus H1 or human papillomavirus.

<p>A: HeLa cells were transfected with siRNAs targeting Ubc9 or Sae2. 46 h later, the cells were infected with ss-firefly luciferase vectors of AAV 1, 8, 9 and capsid variants thereof (left part) and scAAV5- or scAAV9-renilla luciferase (right part) at an MOI of 10⁴. The variants of AAV 1, 8, and 9 harbored heptamer insertions at the threefold spikes in position corresponding to amino acid 588 of AAV2. NYS: Peptide NYSRGVD; NEA: peptide NEAVRE. 25 h after infection, cells were lysed and analyzed for luciferase activity. The RLU values in the case of transfection of the control siRNA ‘AllStars negative control siRNA’ were set to 100. The mean values and standard deviation of three independent experiments are shown. B; C; D: HeLa cells were transfected with siRNA targeting Ubc9 (B) or Sae2 (C and D) 48 h before they were transduced with different recombinant vectors encoding luciferase reporters. Luciferase activity was determined 24 h post infection. The graphs show the ratio of luciferase activity of cells treated with siRNAs targeting Ubc9 or Sae2, respectively and cells treated with <i>AllStars negative control siRNA</i> (scrambled). Shown are the mean of three independent experiments with standard deviations.</p

Effect of SUMOylation pathway on AAV transduction is independent of MOI.

<p>HeLa cells were transfected with different siRNAs and 48 h later transduced with ssAAV-firefly luciferase vectors at different MOIs as indicated. A: relative light units after 24 h incubation determined in three independent experiments, B: ratio of luciferase activity of cells treated with siRNA targeting Ubc9 and cells treated with ‘AllStars negative control siRNA’ (scrambled).</p

Screen of two genome-wide siRNA libraries reveal that the SUMOylation pathway controls AAV transduction.

<p>(a) Distribution of z-scores. A total of 20,290 genes are shown. The dashed lines indicate a z-score threshold of +/- 1.7 resulting in a total of 921 hits, consisting of 740 putative host cell restriction factors (HRF; z-score > 1.7) and 181 putative host cell dependency factors (HDF; z-score < -1.7). Hits concerning gene products of the SUMOylation pathway are indicated. (b) SUMOylation pathway factors and their z-scores identified in the screen are indicated (adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005281#ppat.1005281.ref076" target="_blank">76</a>])</p

Components of the SUMOylation pathway identified as putative AAV host cell restriction factors.

<p>The screening of the genome-wide siRNA library revealed several factors of the SUMOylation pathway influencing AAV transduction. Protein and gene names are indicated. The screens were carried out in duplicates and each gene was targeted by three different siRNAs. The screen identified 740 putative host cell restriction factors, the table shows the ranking of the factors according to their z-score. Note: in the manuscript the protein identifiers are also used in reference to the corresponding gene.</p

Knockdown of either the E1 or E2 enzymes of the SUMOylation pathway results in an increased infection efficiency of scAAV2-eGFP.

<p>HeLa cells were transfected with four different siRNAs targeting expression of Ubc9 and Sae2, respectively. Forty-eighth later, the cells were infected with scAAV2-eGFP at an MOI_GC of 10³. Twenty-four h after infection, half of the cells were proceeded for FACS-analysis, the other half analyzed by western blotting for protein levels after siRNA transfections. (a) Relative MFIs of three independent experiments are shown. The MFI was normalized by setting siRNA transfections with ‘AllStars negative control siRNA’ (Qiagen, indicated as ‘scrambled; scr.’) to 100. (b) The percentage of GFP-positive cells representing infected cells of three independent experiments are shown. (c,d) Western blot analysis showing a reduced steady-state level of Sae2 and Ubc9, respectively, after transfection of the four different siRNAs in comparison to cellular actin. The blot shows whole-cell extracts of two independent transfections for each siRNA. (e) Fluorescence of HeLa cells transduced with scAAV2-GFP vectors. Knockdown of Sae2 or Ubc9 was performed 48 h before transduction, respectively.</p