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Hepatitis E virus (HEV) is a major public health problem with limited therapeutic options. Here, we engineered adeno-associated viral vectors of serotype 6 (AAV6) to express short hairpin RNAs (shRNAs) against HEV transcripts with the prospect of down-regulating HEV replication in vivo. We designed 20 different shRNAs, targeting the genome of the HEV genotype 3 (GT3) Kernow-C1 p6 strain, for delivery upon AAV6 transduction. Using an original selectable HEV GT3 reporter replicon, we identified three shRNAs that efficiently down-regulated HEV replication. We further confirmed their inhibitory potency with full-length HEV infection. Seventy-two hours following transduction, HEV replication in both systems decreased by up to 95%. The three most potent inhibitory shRNAs identified were directed against the methyltransferase domain, the junction region between the open reading frames (ORFs), and the 3´ end of ORF2. Targeting all three regions by multiplexing the shRNAs further enhanced their inhibitory potency over a prolonged period of up to 21 days following transduction. Conclusion: Combining RNA interference and AAV vector-based gene therapy has great potential for suppressing HEV replication. Our strategy to target the viral RNA with multiplexed shRNAs should help to counteract viral escape through mutations. Considering the widely documented safety of AAV vector-based gene therapies, our approach is, in principle, amenable to clinical translation.

Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

Scientific reports 7(1), 2265 (2017). doi:10.1038/s41598-017-02357-0, Published by Nature Publishing Group, London

Localization microscopy approaches allowing an optical resolution down to the single-molecule level in fluorescence-labeled biostructures have already found a variety of applications in cell biology, as well as in virology. Here, we focus on some perspectives of a special localization microscopy embodiment, spectral precision distance/position determination microscopy (SPDM). SPDM permits the use of conventional fluorophores or fluorescent proteins together with standard sample preparation conditions employing an aqueous buffered milieu and typically monochromatic excitation. This allowed superresolution imaging and studies on the aggregation state of modified tobacco mosaic virus particles on the nanoscale with a single-molecule localization accuracy of better than 8 nm, using standard fluorescent dyes in the visible spectrum. To gain a better understanding of cell entry mechanisms during influenza A virus infection, SPDM was used in conjunction with algorithms for distance and cluster analyses to study changes in the distribution of virus particles themselves or in the distribution of infection-related proteins, the hepatocyte growth factor receptors, in the cell membrane on the single-molecule level. Not requiring TIRF (total internal reflection) illumination, SPDM was also applied to study the molecular arrangement of gp36.5/m164 glycoprotein (essentially associated with murine cytomegalovirus infection) in the endoplasmic reticulum and the nuclear membrane inside cells with single-molecule resolution. On the basis of the experimental evidence so far obtained, we finally discuss additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligonucleotide fluorescence in situ hybridization, as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices.

The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples provides can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular sub-populations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.Abbreviations3D-TIM3D targeted imagingALTalternative lengthening of telomeresAPBALT-associated PML-NBCLSMconfocal laser scanning fluorescence microscopyECTRextrachromosomal telomeric repeatFFPEformalin-fixed, paraffin-embeddedFISHfluorescence in situ hybridizationIFImmunofluorescencepedGBMpediatric glioblastomaPMLpromyelocytic leukemiaPML-NBPML nuclear bodyPNApeptide nucleic acidROIregion of interestTMAtissue microarrayTMMtelomere maintenance mechanismSMLMsingle molecule localization microscopy

A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.

International audience; The Dual Color Localization Microscopy (2CLM) presented here is based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorochromes under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length-scales of 30 nm.

The copy number of a specific mRNA and its subcellular localization in cells is a mode by which cells can regulate protein synthesis. Beta-actin mRNA is transported to filopodia upon serum stimulation, regions of cell growth and movement where actin polymerization is essential (1). The copy number of mRNA molecules directly translates to the amount of actin monomers that are synthesized. Quantifying the actin mRNA copy numbers provides a measure of how polymerization of actin is regulated.To this extent, we apply stochastic super-resolution microscopy with photoswitchable fluorescent probes combined with single-molecule localization (2,3) for quantitative studies of mRNA concentration and localization in eukaryotic cells. For this purpose, we apply various labelling approaches for mRNA, including well-established methods such as fluorescence in-situ hybridization (FISH) (4). Focussing on actin mRNA, we derive quantitative information on copy numbers, and we apply coordinate-based algorithms to analyze our single-molecule super-resolution data.References(1) Ben-Ari et al. (2010) J Cell Sci, 123, 1761-1774.(2) Betzig et al. (2006) Science, 313, 1642-1645.(3) Heilemann et al. (2008) Angew. Chemie, 47, 6172-6176.(4) Levskey, J. M.; Singer, R. (2003) Journal of Cell Science, 116, 2833-2838.