Glycosylation of recombinant adeno-associated virus serotype 6.

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galß1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products.

Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly.

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

A G-quadruplex structure at the 5' end of the H19 coding region regulates H19 transcription.

The H19 gene, one of the best known imprinted genes, encodes a long non-coding RNA that regulates cell proliferation and differentiation. H19 RNA is widely expressed in embryonic tissues, but its expression is restricted in only a few tissues after birth. However, regulation of H19 gene expression remains poorly understood outside the context of genomic imprinting. Here we identified evolutionarily conserved guanine (G)-rich repeated motifs at the 5' end of the H19 coding region that are consistent with theoretically deduced G-quadruplex sequences. Circular dichroism spectroscopy and electrophoretic mobility shift assays with G-quadruplex-specific ligands revealed that the G-rich motif, located immediately downstream of the transcription start site (TSS), forms a G-quadruplex structure in vitro. By using a series of mutant forms of H19 harboring deletion or G-to-A substitutions, we found that the H19-G-quadruplex regulates H19 gene expression. We further showed that transcription factors Sp1 and E2F1 were associated with the H19-G-quadruplex to either suppress or promote the H19 transcription, respectively. Moreover, H19 expression during differentiation of mouse embryonic stem cells appears to be regulated by a genomic H19 G-quadruplex. These results demonstrate that the G-quadruplex structure immediately downstream of the TSS functions as a novel regulatory element for H19 gene expression.

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

Quantification of the dynamics of enterovirus 71 infection by experimental-mathematical investigation.

Enterovirus 71 (EV71) is the causative agent of hand-foot-and-mouth disease and can trigger neurological disorders. EV71 outbreaks are a major public health concern in Asia-Pacific countries. By performing experimental-mathematical investigation, we demonstrate here that viral productivity and transmissibility but not viral cytotoxicity are drastically different among EV71 strains and can be associated with their epidemiological backgrounds. This is the first report demonstrating the dynamics of nonenveloped virus replication in cell culture using mathematical modeling.

Vpu augments the initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in humanized mice.

While human cells express potent antiviral proteins as part of the host defense repertoire, viruses have evolved their own arsenal of proteins to antagonize them. BST2 was identified as an inhibitory cellular protein of HIV-1 replication, which tethers virions to the cell surface to prevent their release. On the other hand, the HIV-1 accessory protein, Vpu, has the ability to downregulate and counteract BST2. Vpu also possesses the ability to downmodulate cellular CD4 and SLAMF6 molecules expressed on infected cells. However, the role of Vpu in HIV-1 infection in vivo remains unclear. Here, using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrate that Vpu contributes to the efficient spread of HIV-1 in vivo during the acute phase of infection. Although Vpu did not affect viral cytopathicity, target cell preference, and the level of viral protein expression, the amount of cell-free virions in vpu-deficient HIV-1-infected mice was profoundly lower than that in wild-type HIV-1-infected mice. We provide a novel insight suggesting that Vpu concomitantly downregulates BST2 and CD4, but not SLAMF6, from the surface of infected cells. Furthermore, we show evidence suggesting that BST2 and CD4 impair the production of cell-free infectious virions but do not associate with the efficiency of cell-to-cell HIV-1 transmission. Taken together, our findings suggest that Vpu downmodulates BST2 and CD4 in infected cells and augments the initial burst of HIV-1 replication in vivo. This is the first report demonstrating the role of Vpu in HIV-1 infection in an in vivo model.