Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes

Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.     

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.     

LKB1 loss in melanoma disrupts directional migration toward extracellular matrix cues

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility. Using a microfluidic gradient chamber system and time-lapse microscopy, in this paper, we uncover a new function for LKB1 as a directional migration sensor of gradients of extracellular matrix (haptotaxis) but not soluble growth factor cues (chemotaxis). Systematic perturbation of known LKB1 effectors demonstrated that this response does not require canonical adenosine monophosphate–activated protein kinase (AMPK) activity but instead requires the activity of the AMPK-related microtubule affinity-regulating kinase (MARK)/PAR-1 family kinases. Inhibition of the LKB1–MARK pathway facilitated invasive motility, suggesting that loss of the ability to sense inhibitory matrix cues may promote melanoma invasion.     

Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo

A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/α1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8⁺ CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.Adeno-associated virus (AAV) is a single-stranded DNA parvovirus. Its replication relies on coinfection of a helper virus such as adenovirus or herpesvirus. In the absence of a helper virus, AAV establishes latency to integrate into the AAVS1 site of host chromosome 19 (11). The genome of AAV is ∼4.7 kb and contains two open reading frames encoding replication proteins and structural capsid proteins (21). The capsid proteins (VP) are composed of VP1, VP2, and VP3. The VP3 protein is the major structural component and constitutes nearly 80% of the virion shell with an overall ratio of 1:1:8 for VP1, VP2, VP3, respectively. While VP2 is thought to be nonessential for AAV transduction (30), the VP1 subunit contains a phospholipase A2 domain required for infectivity (9). Recombinant AAV (rAAV) vectors require only the 145-bp terminal repeats of the AAV genome in cis and all other viral factors supplied in trans for production (18). rAAV vectors have rapidly gained popularity in gene therapy applications and have proven effective in preclinical studies/clinical trials for a number of diseases (20, 31, 33).AAV vectors mount a potent humoral immune response against capsid in animals and human. However, AAV vectors only contain the therapeutic gene flanked by two 145-bp AAV terminal repeats devoid of any AAV genes(23). In addition, AAV initiates long-term stable therapeutic gene expression in animal models (3-5, 17, 31). Based on these observations AAV has been thought to be relatively nonimmunogenic regarding the induction of cytotoxic T lymphocytes (CTLs) specific for capsid proteins. In spite of all of these observations, the recent clinical trial for hemophilia B (F9) gene therapy has otherwise suggested that AAV2 capsid initiates cell-mediated immunity that eliminates the AAV2 encoding F9 (AAV2/F9) vector transduced liver cells (15). Against this backdrop, numerous attempts to replicate aforementioned observations in animal models have been made. Preliminary results from these studies support direct presentation and cross-presentation of the AAV2 capsid in animal models (6, 12, 13, 22, 29). However, capsid-specific CTLs did not eliminate AAV2-transduced target cells in mice (12, 13, 29), inconsistent with observations made in a clinical trial for hemophilia B with AAV2/F9 gene therapy. A potential explanation for this discrepancy is the weak immunogenicity of the AAV2 capsid in mice. Accordingly, we hypothesized that incorporation of a peptide epitope into the AAV2 capsid would increase immunogenicity of the rAAV and therefore could be exploited to mimic events ongoing in humans and study approaches to block capsid-specific CTL reactivity in mice.We chose to introduce the MHC-H2K^b-restricted SIINFEKL peptide derived from ovalbumin (OVA) into AAV2 capsid. Integration of the OVA epitope into AAV capsids elicited a specific CTL response. Most importantly, after administration of genetically engineered AAV2 vectors into OVA peptide-immunized mice, OVA-specific CTL reactivity was further enhanced, thereby limiting transgene expression in vivo. The modified vector described herein is a potentially valuable tool for future studies focused on developing strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in animal models.     

Cloning,Characterization and Diversity of Insecticidal Crystal Protein Genes of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Native Isolates from Soils of Andaman and Nicobar Islands

Bt strains were isolated from soils of Andaman and Nicobar Islands and characterized by microscopic and molecular methods. Diversity was observed both in protein and cry gene profiles, where majority of the isolates showed presence of 65 kDa protein band on SDS-PAGE while rest of them showed 130, 72, 44, and 29 kDa bands. PCR analysis revealed predominance of cry1I and cry7, 8 genes in these isolates. The PCR screening strategy presented here led us to identify putative novel cry genes which could be active against Coleoptera insects. Variation in the nucleotide sequences of cry genes from the isolates suggests that the genetic diversity of Bt isolates results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats in the soils of Andaman and Nicobar islands. The implications of our studies are important from the point of view of identifying novel cry genes that could be toxic to insects other than lepidoptera.     

Prey preference of large carnivores in Anamalai Tiger Reserve,India

Prey preferences of large carnivores (tiger (Panthera tigris), leopard (Panthera pardus) and dhole (Cuon alpinus)) in the tropical forest of Anamalai Tiger Reserve (ATR) were evaluated. This was the first study in ATR to estimate the density of prey and the food habits of these large carnivores. The 958-km² intensive study area was found to have a high mammalian prey density (72.1 animals per square kilometre) with wild boar (20.61 animals per square kilometre) and chital (20.54 animals per square kilometre) being the most common species, followed by nilgiri tahr (13.6 animals per square kilometre). When the density figures were multiplied by the average weight of each prey species, a high biomass density of 14,204 kg km⁻² was obtained for the intensive study area. Scat analysis and incidental kill observation were used to determine the dietary composition of these predators. During the study from the period of March 2001 to April 2004, 1,145 tiger scats, 595 leopard scats and 2,074 dhole scats were collected and analysed. Kill data were based on direct observation of 66 tiger kills and 39 leopard kills. Sambar, with a density of 6.54 kg km⁻² was the preferred prey for these carnivores. Sambar constitutes 35% of the overall diet of tiger, whereas it constitutes 17% and 25% in leopard and dhole diets, respectively. Chital was utilized less than sambar in the range of about 7%, 11% and 15% by tiger, leopard and dhole, respectively. Predator diet was estimated more accurately by scat analysis, which reveals 30% of smaller prey species in leopard’s diet, which was not observed by kill data. This study reveals that ATR harbours high prey density, and these large carnivores seem mostly dependent on the wild prey rather than on domestic livestock as in some other areas in the subcontinent. These factors make ATR a potential area for long-term conservation of these endangered carnivores.     

Bocavirus episome in infected human tissue contains non-identical termini

Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses. Significance: HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.     

Glycated AAV vectors: chemical redirection of viral tissue tropism

A chemical approach for selective masking of arginine residues on viral capsids featuring an exogenous glycation reaction has been developed. Reaction of adeno-associated viral (AAV) capsids with the α-dicarbonyl compound, methylglyoxal, resulted in formation of arginine adducts. Specifically, surface-exposed guanidinium side chains were modified into charge neutral hydroimidazolones, thereby disrupting a continuous cluster of basic amino acid residues implicated in heparan sulfate binding. Consequent loss in heparin binding ability and decrease in infectivity were observed. Strikingly, glycated AAV retained the ability to infect neurons in the mouse brain and were redirected from liver to skeletal and cardiac muscle following systemic administration in mice. Further, glycated AAV displayed altered antigenicity demonstrating the potential for evading antibody neutralization. Generation of unnatural amino acid side chains through capsid glycation might serve as an orthogonal strategy to engineer AAV vectors displaying novel tissue tropisms for gene therapy applications.