Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen

2,3 and 2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection

Evolution of dependoparvoviruses across geological timescales – implications for design of AAV-based gene therapy vectors

Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyze the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha (whales and hippos), Vespertillionade (smooth-nosed bats), and Lagomorpha (rabbits, hares, and pikas), indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77-23 million years ago (MYA). These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously-replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modeling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions (VRs), especially in those related to the threefold and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 (PLA2) domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus “fossils” relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based upon paleovirology data

Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested

Characterization of carbonic anhydrase isozyme specific inhibition by sulfamated 2-ethylestra compounds

Sulfamated 2-ethylestra compounds have demonstrated strong anticancer activity, high bioavailability and an ability to bypass liver metabolism by reversibly binding carbonic anhydrase (CA) II in erythrocytes. Another CA isoform, CA IX, is overexpressed in many cancers. The CA domain of CA IX is oriented extracellularly, which may permit targeting inhibitors to tumors. Presented here is the characterization of three 2-ethylestra compounds bound to both CA II and a CA IX mimic protein. Inhibition by 18O exchange showed that compound 16 demonstrated an approximately 12-fold higher affinity for CA II over CA IX mimic. Structurally, compounds 15 and 16 showed alternate binding modes between CA II and CA IX mimic, apparently due to a water-mediated hydrogen bond to the isozyme-specific residue 67. Though the specificity was demonstrated for CA II over CA IX, this study reveals insights that may be key to developing isozyme specific CA inhibitors for novel anticancer therapies.This work was supported by NIH Grant GM25154 and by grants from the Medical Research Council of South Africa (AG374, AK076), the Cancer Association of South Africa (AK246), the Struwig-Germeshuysen Cancer Research Trust of South Africa (AJ038) and RESCOM University of Pretoria (A0R984).http://www.benthamscience.com/lddd/nf201

Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. To tackle this problem, we developed a structure-guided approach to evolve AAV variants with altered antigenic footprints that cannot be recognized by preexisting antibodies. These proof-of-principle studies demonstrate that synthetic AAV variants can be evolved to evade neutralizing sera from different species—mice, nonhuman primates, and humans—without compromising yield and transduction efficiency or altering tropism. Our approach provides a roadmap for engineering any AAV strain to evade NAbs in prospective patients for human gene therapy

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration