Cryo-EM structure of adeno-associated virus 4 at 2.2†Šresolution.

Adeno-associated virus (AAV) is the vector of choice for several approved gene-therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, a high-resolution cryo-electron microscopy structure (2.2 Å) of AAV serotype 4 (AAV4) is presented. The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, AAV12 and AAVrh32.33) is unknown. Other AAVs interact with the same cell receptor, adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like 1 (PKD1) domain of AAVR, while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2 of AAVR. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear to be sufficient to exclude binding with PKD1.

Adeno-Associated Virus Receptor-Binding: Flexible Domains and Alternative Conformations through Cryo-Electron Tomography of Adeno-Associated Virus 2 (AAV2) and AAV5 Complexes.

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now ß-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the ßA-ßB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry.

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.

The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors.

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli.

Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 (AAV-2) and (b) two fragments from the binding ectodomain of AAV's cellular receptor, AAVR. His6PKD1-2 comprises the first two polycystic kidney disease (PKD) domains, the minimal required for efficient binding of AAV, expressed with an N-terminal histidine tag. MBP-PKD1-5 is a fusion of the maltose binding protein with all five of the PKD domains of the AAVR receptor. Presented are the expression and purification of milligram quantities, ample for in vitro analyses. For AAV-2, the protocol offers an alternative to the use of (infectious) wild-type virus or transducing vectors. One of the methods for producing transducing vector is in Sf9 cells, and the production of VLPs is based on this. For AAVR, the protocols enable biochemical and biophysical characterization of virus-binding. The minimal two-domain construct allows more saturated binding to symmetry-equivalent sites on the virus, while the larger construct might be better expected to reflect the native receptor.

Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle.

Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO4 2- ), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment.

Cr(VI) reduction and physiological toxicity are impacted by resource ratio in Desulfovibrio vulgaris.

Desulfovibrio spp. are capable of heavy metal reduction and are well-studied systems for understanding metal fate and transport in anaerobic environments. Desulfovibrio vulgaris Hildenborough was grown under environmentally relevant conditions (i.e., temperature, nutrient limitation) to elucidate the impacts on Cr(VI) reduction on cellular physiology. Growth at 20 °C was slower than 30 °C and the presence of 50 µM Cr(VI) caused extended lag times for all conditions, but once growth resumed the growth rate was similar to that without Cr(VI). Cr(VI) reduction rates were greatly diminished at 20 °C for both 50 and 100 µM Cr(VI), particularly for the electron acceptor limited (EAL) condition in which Cr(VI) reduction was much slower, the growth lag much longer (200 h), and viability decreased compared to balanced (BAL) and electron donor limited (EDL) conditions. When sulfate levels were increased in the presence of Cr(VI), cellular responses improved via a shorter lag time to growth. Similar results were observed between the different resource (donor/acceptor) ratio conditions when the sulfate levels were normalized (10 mM), and these results indicated that resource ratio (donor/acceptor) impacted D. vulgaris response to Cr(VI) and not merely sulfate limitation. The results suggest that temperature and resource ratios greatly impacted the extent of Cr(VI) toxicity, Cr(VI) reduction, and the subsequent cellular health via Cr(VI) influx and overall metabolic rate. The results also emphasized the need to perform experiments at lower temperatures with nutrient limitation to make accurate predictions of heavy metal reduction rates as well as physiological states in the environment.

Deconstructing the Dissimilatory Sulfate Reduction Pathway: Isotope Fractionation of a Mutant Unable of Growth on Sulfate.

The sulfur isotope record provides key insight into the history of Earth's redox conditions. A detailed understanding of the metabolisms driving this cycle, and specifically microbial sulfate reduction (MSR), is crucial for accurate paleoenvironmental reconstructions. This includes a precise knowledge of the step-specific sulfur isotope effects during MSR. In this study, we aim at resolving the cellular-level fractionation factor during dissimilatory sulfite reduction to sulfide within MSR, and use this measured isotope effect as a calibration to enhance our understanding of the biochemistry of sulfite reduction. For this, we merge measured isotope effects associated with dissimilatory sulfite reduction with a quantitative model that explicitly links net fractionation, reaction reversibility, and intracellular metabolite levels. The highly targeted experimental aspect of this study was possible by virtue of the availability of a deletion mutant strain of the model sulfate reducer Desulfovibrio vulgaris (strain Hildenborough), in which the sulfite reduction step is isolated from the rest of the metabolic pathway owing to the absence of its QmoABC complex (ΔQmo). This deletion disrupts electron flux and prevents the reduction of adenosine phosphosulfate (APS) to sulfite. When grown in open-system steady-state conditions at 10% maximum growth rate in the presence of sulfite and lactate as electron donor, sulfur isotope fractionation factors averaged -15.9‰ (1 σ = 0.4), which appeared to be statistically indistinguishable from a pure enzyme study with dissimilatory sulfite reductase. We coupled these measurements with an understanding of step-specific equilibrium and kinetic isotope effects, and furthered our mechanistic understanding of the biochemistry of sulfite uptake and ensuing reduction. Our metabolically informed isotope model identifies flavodoxin as the most likely electron carrier performing the transfer of electrons to dissimilatory sulfite reductase. This is in line with previous work on metabolic strategies adopted by sulfate reducers under different energy regimes, and has implications for our understanding of the plasticity of this metabolic pathway at the center of our interpretation of modern and palaeo-environmental records.

Effect of the deletion of qmoABC and the promoter-distal gene encoding a hypothetical protein on sulfate reduction in Desulfovibrio vulgaris Hildenborough.

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5'-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the Delta(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.