Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry.

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and MS. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the Escherichia coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extracts, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extracts, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid-binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

Investigating Daptomycin-Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs.

Daptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin-membrane interactions with different lipid bilayer nanodiscs. Native MS suggests that daptomycin incorporates randomly and does not prefer any specific oligomeric states when integrated into bilayers. FPOP reveals significant protection in most bilayer environments. Combining the native MS and FPOP results, we observed that stronger membrane interactions are formed with more rigid membranes, and pore formation may occur in more fluid membranes to expose daptomycin to FPOP oxidation. Electrophysiology measurements further supported the observation of polydisperse pore complexes from the MS data. Together, these results demonstrate the complementarity of native MS, FPOP, and membrane conductance experiments to shed light on how antibiotic peptides interact with and within lipid membranes.

Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry.

Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.

Quantification of intracellular HNO delivery with capillary zone electrophoresis.

Redox signaling, wherein reactive and diffusible small molecules are channeled into specific messenger functions, is a critical component of signal transduction. A central principle of redox signaling is that the redox modulators are produced in a highly controlled fashion to specifically modify biotargets. Thiols serve as primary mediators of redox signaling as a function of the rich variety of adducts, which allows initiation of distinct cellular effects. Coupling the inherent reactivity of thiols with highly sensitive and selective chemical analysis protocols can facilitate identification of redox signaling agents, both in solution and in cultured cells. Here, we describe use of capillary zone electrophoresis to both identify and quantify sulfinamides, which are specific markers of the reaction of thiols with nitroxyl (HNO), a putative biologically relevant reactive nitrogen species.

Nanomechanical Properties of Artificial Lipid Bilayers Composed of Fluid and Polymerizable Lipids.

Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (Langmuir, 2019, 35, 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains. Here we used atomic force microscopy (AFM) to compare the nanoscale mechanical properties of these binary PSLBs with single-component PSLBs. The elastic (Young's) modulus, area compressibility modulus, and bending modulus of bis-SorbPC PSLBs increased upon polymerization. Before polymerization, breakthrough events at forces below 5 nN were observed, but after polymerization, the AFM tip could not penetrate the PSLB up to an applied force of 20 nN. These results are attributed to the polymeric network in poly(bis-SorbPC), which increases the bilayer stiffness and resists compression and bending. In binary DPhPC/poly(bis-SorbPC) PSLBs, the DPhPC domains are less stiff, more compressible, and are less resistant to rupture and bending compared to pure DPhPC bilayers. These differences are attributed to bis-SorbPC monomers and oligomers present in DPhPC domains that disrupt the packing of DPhPC molecules. In contrast, the poly(bis-SorbPC) domains are stiffer and less compressible relative to pure PSLBs; this difference is attributed to DPhPC filling the nm-scale pores in the polymerized domains that are created during bis-SorbPC polymerization. Thus, incomplete phase segregation increases the stability of poly(bis-SorbPC) but has the opposite, detrimental effect for DPhPC. Overall, these results provide guidance for the design of partially polymerized bilayers for technological uses.

Direct interaction of the ATP-sensitive K+ channel by the tyrosine kinase inhibitors imatinib, sunitinib and nilotinib.

The ATP-regulated K+ channel (KATP) plays an essential role in the control of many physiological processes, and contains a ATP-binding site. Tyrosine kinase inhibitors (TKI) are commonly used drugs, that primarily target ATP-binding sites in tyrosine kinases. Herein, we used the patch-clamp technique to examine the effects of three clinically established TKIs on KATP channel activity in isolated membrane patches, using a pancreatic ß-cell line as a KATP channel source. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by imatinib with half-maximal concentration of approximately 9.4 µM. The blocking effect of imatinib was slow and reversible. No effect of imatinib was observed on either the large (KBK) or the small (KSK) conductance, Ca2+-regulated K+ channel. In the presence of ATP/ADP (ratio 1) addition of imatinib increased channel activity approximately 1.5-fold. Sunitinib and nilotinib were also found to decrease KATP channel activity. These findings are compatible with the view that TKIs, designed to interact at the ATP-binding pocket on the tyrosine receptor, also interact at the ATP-binding site on the KATP channel. Possibly, this might explain some of the side effects seen with TKIs.

Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids.

Polymerization of synthetic phospholipid monomers has been widely used to enhance the stability of lipid membranes in applications such as membrane-based biosensing, where the inherent instability of fluid-phase lipid bilayers can be problematic. However, lipid polymerization typically decreases membrane fluidity, which may be required to maintain the activity of reconstituted integral proteins and peptides. Prior work has shown that a bilayer composed of binary mixtures of poly(lipid) and fluid lipid exhibits enhanced stability and supports the function of incorporated biomolecules. This work examines the structural basis of these findings using planar supported lipid bilayers (PSLBs) composed of binary mixtures of a polymerizable lipid, 1,2-bis[10-(2',4'-hexadienoloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC), and a nonpolymerizable lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Fluorescence recovery after photobleaching (FRAP) measurements showed that long-range lateral diffusion was minimally affected when the poly(lipid) mole ratio was ≤0.7. Atomic force microscopy, used to examine phase segregation in these PSLBs, showed that DPhPC forms a continuous lipid matrix that is 0.2-0.4 nm thicker than the island-like poly(bis-SorbPC) domains, with lateral dimensions of ≤200 nm. The nanoscale phase segregation allows for long-range lateral diffusion of lipid probes in the DPhPC matrix. The combination of fluidity and stability in these materials should make them useful in membrane-based biosensing applications.

Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ±â€¯3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ±â€¯25 µM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ±â€¯0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ±â€¯0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

Enhanced Temporal Resolution with Ion Channel-Functionalized Sensors Using a Conductance-Based Measurement Protocol.

The binding of a target analyte to an ion channel (IC), which is readily detected electrochemically in a label-free manner with single-molecule selectivity and specificity, has generated widespread interest in using natural and engineered ICs as transducers in biosensing platforms. To date, the majority of developments in IC-functionalized sensing have focused on IC selectivity or sensitivity or development of suitable membrane environments and aperture geometries. Comparatively little work has addressed analytical performance criteria, particularly criteria required for temporal measurements of dynamic processes. We report a measurement protocol suitable for rapid, time-resolved monitoring (≤30 ms) of IC-modulated membrane conductance. Key features of this protocol include the reduction of membrane area and the use of small voltage steps (10 mV) and short duration voltage pulses (10 ms), which have the net effect of reducing the capacitive charging and decreasing the time required to achieve steady state currents. Application of a conductance protocol employing three sequential, 10 ms voltage steps (-10 mV, -20 mV, -30 mV) in an alternating, pyramid-like arrangement enabled sampling of membrane conductance every 30 ms. Using this protocol, dynamic IC measurements on black lipid membranes (BLMs) functionalized with gramicidin A were conducted using a fast perfusion system. BLM conductance decreased by 76 ± 7.5% within 30 ms of switching from solutions containing 0 to 1 M Ca2+, which demonstrates the feasibility of using this approach to monitor rapid, dynamic chemical processes. Rapid conductance measurements will be broadly applicable to IC-based sensors that undergo analyte-specific gating.

Emerging trends in precision fabrication of microapertures to support suspended lipid membranes for sensors, sequencing, and beyond.

Suspended lipid membranes, also called black lipid membranes (BLMs), are an important model system that approximates the lipid bilayer environment of cell membranes. Increasingly, BLMs are utilized in sensing strategies that harness high sensitivity measurements of ion flux across the membrane, typically facilitated by ion channel proteins. BLMs are suspended across microapertures that connect two otherwise isolated fluidic compartments, and the precision fabrication of such microapertures can contribute to the stability and performance of the resulting BLM. Here, we highlight two emerging trends in the precision fabrication of microapertures for BLM formation: microfabrication in silicon-based thin film substrates, and microfabrication in the negative photoresist material SU-8. Four unique fabrication strategies are outlined, and we project the impact that these microfabrication strategies will have for BLM-integrated bioanalytical technologies.

Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis.

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.

Ion channel probes for scanning ion conductance microscopy.

The sensitivity and selectivity of ion channels provide an appealing opportunity for sensor development. Here, we describe ion channel probes (ICPs), which consist of multiple ion channels reconstituted into lipid bilayers suspended across the opening of perflourinated glass micropipets. When incorporated with a scanning ion conductance microscope (SICM), ICPs displayed a distance-dependent current response that depended on the number of ion channels in the membrane. With distance-dependent current as feedback, probes were translated laterally, to demonstrate the possibility of imaging with ICPs. The ICP platform yields several potential advantages for SICM that will enable exciting opportunities for incorporation of chemical information into imaging and for high-resolution imaging.

Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases.

Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.

Evidence for Ca(2+)-regulated ATP release in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca(2+)-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca(2+) influx since exclusion of extracellular Ca(2+) diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

Transplantation and Noninvasive Longitudinal In Vivo Imaging of Parathyroid Cells: A Proof-of-Concept Study.

The parathyroid cell is a vital regulator of extracellular calcium levels, operating through the secretion of parathyroid hormone (PTH). Despite its importance, the regulation of PTH secretion remains complex and not fully understood, representing a unique interplay between extracellular and intracellular calcium, and hormone secretion. One significant challenge in parathyroid research has been the difficulty in maintaining cells ex vivo for in-depth cellular investigations. To address this issue, we introduce a novel platform for parathyroid cell transplantation and noninvasive in vivo imaging using the anterior chamber of the eye as a transplantation site. We found that parathyroid adenoma tissue transplanted into the mouse eye engrafted onto the iris, became vascularized, and retained cellular composition. Transplanted animals exhibited elevated PTH levels, indicating a functional graft. With in vivo confocal microscopy, we were able to repetitively monitor parathyroid graft morphology and vascularization. In summary, there is a pressing need for new methods to study complex cellular processes in parathyroid cells. Our study provides a novel approach for noninvasive in vivo investigations that can be applied to understand parathyroid physiology and pathology under physiological and pathological conditions. This innovative strategy can deepen our knowledge on parathyroid function and disease.

Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies.

Robust and high-density biosensors incorporating suspended lipid membranes require microfabricated apertures that can be readily integrated into complex analysis systems. Apertures with well-defined, three-dimensional geometries enable the formation of suspended lipid membranes and facilitate reduced aperture size compared to vertical-walled apertures. Unfortunately, existing methods of producing apertures with well-defined, three-dimensional geometries are based on complex and expensive fabrication procedures, some of which yield apertures in excessively fragile thin-film materials. Here, we describe a microfabrication method utilizing incline and rotate lithography that achieves sloped-wall microapertures in SU-8 polymer substrates with precision control of the aperture diameter, substrate thickness, and wall angle. This approach is simple, is of low cost, and is readily scaled up to allow highly reproducible parallel fabrication. The effect of the incident angle of UV exposure and the size of photomask features on the aperture geometry were investigated, yielding aperture diameters as small as 7 µm and aperture wall angles ranging from 8° to 36° measured from the normal axis. Black lipid membranes were suspended across the apertures and showed normalized conductance values of 0.02-0.05 pS µm(-2) and breakdown voltages of 400-600 mV. The functionality of the resulting sloped-wall microapertures was validated via measurement of reconstituted α-hemolysin activity and the voltage-gated channel activity of alamethicin.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods.

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for the correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similarities, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainty in distinguishing empty and filled capsids. To address these challenges, we combined two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, a selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids. Liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were then used for the capsid denaturing measurements. This multimethod combination was applied to three AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.