Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I.

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single-stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas-phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.

Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes.

After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

Liposomal leakage induced by virus-derived peptides, viral proteins, and entire virions: rapid analysis by chip electrophoresis.

Permeabilization of model lipid membranes by virus-derived peptides, viral proteins, and entire virions of human rhinovirus was assessed by quantifying the release of a fluorescent dye from liposomes via a novel chip electrophoretic assay. Liposomal leakage readily occurred upon incubation with the pH-sensitive synthetic fusogenic peptide GALA and, less efficiently, with a 24mer peptide (P1-N) derived from the N-terminus of the capsid protein VP1 of human rhinovirus 2 (HRV2) at acidic pH. Negative stain transmission electron microscopy showed that liposomes incubated with the rhinovirus-derived peptide remained largely intact. At similar concentrations, the GALA peptide caused gross morphological changes of the liposomes. On a molar basis, the leakage-inducing efficiency of the P1 peptide was by about 2 orders of magnitude inferior to that of recombinant VP1 (from HRV89) and entire HRV2. This underscores the role in membrane destabilization of VP1 domains remote from the N-terminus and the arrangement of the peptide in the context of the icosahedral virion. Our method is rapid, requires tiny amounts of sample, and allows for the parallel determination of released and retained liposomal cargo.

Chip electrophoretic characterization of liposomes with biological lipid composition: Coming closer to a model for viral infection.

In first attempts at elucidating the transfer of the RNA genome of a human Rhinovirus through lipid membranes in vitro we made use of liposomes decorated with recombinant receptors. This model system was characterized previously by CE but suffered from the requirement for inclusion of polyethylene glycol-modified lipids for reliable separations [Weiss, V. U., Bilek, G., Pickl-Herk, A., Blaas, D., Kenndler, E., Electrophoresis 2009, 30, 2123-2128.]. We here report the analysis of liposomes with a lipid composition much more similar to that of biological lipid bilayers. We found that vesicles containing and lacking this non-physiologic lipid differ significantly in their electrophoretic mobility (by factor 2) although the concentration of charge-bearing lipids in their bilayers is the same. We demonstrate that binding of a human Rhinovirus to the latter liposomes decorated with a cognate receptor can be analysed via electrophoresis on microchips; we support our results with transmission electron microscopy.

Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection.

Minor group human rhinoviruses (HRVs) bind three members of the low-density lipoprotein receptor (LDLR) family: LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP). Whereas ICAM-1, the receptor of major group HRVs actively contributes to viral uncoating, LDLRs are rather considered passive vehicles for cargo delivery to the low-pH environment of endosomes. Since the Tyr-Trp-Thr-Asp beta-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL via intramolecular competition at low pH, we studied whether it also plays a role in HRV infection. Human cell lines deficient in LDLR family proteins are not available. Therefore, we used CHO-ldla7 cells that lack endogenous LDLR. These were stably transfected to express either wild-type (wt) human LDLR or a mutant with a deletion of the beta-propeller. When HRV2 was attached to the propeller-negative LDLR, a lower pH was required for conversion to subviral particles than when attached to wt LDLR. This indicates that high-avidity receptor binding maintains the virus in its native conformation. HRV2 internalization directed the mutant LDLR but not wt LDLR to lysosomes, resulting in reduced plasma membrane expression of propeller-negative LDLR. Infection assays using a CHO-adapted HRV2 variant showed a delay in intracellular viral conversion and de novo viral synthesis in cells expressing the truncated LDLR. Our data indicate that the beta-propeller attenuates the virus-stabilizing effect of LDLR binding and thereby facilitates RNA release from endosomes, resulting in the enhancement of infection. This is a nice example of a virus exploiting high-avidity multimodule receptor binding with an intrinsic release mechanism.

Mimicking virus attachment to host cells employing liposomes: analysis by chip electrophoresis.

Electrophoresis on a chip increasingly replaces electrophoresis in the capillary format because of its speed and containment of the sample within a disposable cartridge. In this paper we demonstrate its utility in the analysis of the interaction between a virus and a liposome-anchored receptor, mimicking viral attachment to host cells. This became possible because detergents, obligatory constituents of the BGE for capillary electrophoretic separation of the virus, were not necessary in the chip format. Separations were carried out in sodium borate buffer, pH 8.3. Liposomes and virus were both labeled for laser-induced fluorescence detection at lambda(ex)/lambda(em) 630/680 nm. Free virus and virus-receptor complexes were resolved from virus attached to receptor-decorated liposomes in the absence of additives or sieving matrices within about 30 s on commercially available microfluidic chips.

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins.

The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N-terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH 8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation.

Capillary electrophoresis of viruses, subviral particles and virus complexes.

CZE and CIEF were so far applied to the analysis of tobacco mosaic virus, Semliki forest virus, human rhinovirus, adenovirus, norovirus and the bacteriophages T5 and MS2. The concentration of viral or subviral particles, of capsid proteins and viral genomes were determined, their electrophoretic mobilities and pI values were measured and bioaffinity reactions between viruses and antibodies, antibody fragments and receptor fragments were assessed. The role of detergents added to the BGE to obtain reproducible electrophoretic conditions was elucidated. The analytes were detected via their UV-absorbance or via fluorescence after derivatization of the viral capsid, the nucleic acid, or both. A new dimension to the detection is added by the possibility of making use of the viral infectivity. At least in theory, this allows for the unequivocal identification of a single infectious virus particle after collection at the capillary outlet. This review summarizes the 25 papers so far published on this topic.

Mimicking early events of virus infection: capillary electrophoretic analysis of virus attachment to receptor-decorated liposomes.

The attachment of human rhinovirus serotype 2 to an artificial cell membrane was followed by capillary electrophoresis. The cell membrane was mimicked by liposomes (average diameter of about 190 nm) containing a lipid with a nitrilotriacetic acid (NTA) group. This group, in the presence of Ni(2+) ions, served as anchor for the his(6)-tags of recombinant derivatives of the very-low-density lipoprotein (VLDL) receptor comprising either modules 1, 2, and 3 (V123) or five tandem copies of module 3 (V33333). We demonstrate by capillary electrophoresis with laser induced fluorescence detection of the liposomes that the minor receptor group rhinovirus HRV2 binds specifically to the receptor-decorated vesicles; the major receptor group rhinovirus HRV14, which uses a different receptor for cell binding, does not attach to the liposomes.

Capillary electrophoresis of liposomes functionalized for protein binding.

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.

Analysis of liposomes by capillary electrophoresis and their use as carrier in electrokinetic chromatography.

This contribution reviews work about liposomes in the context of electrically driven separation methods in the capillary format. The discussion covers four topics. The one broaches the application of liposomes as pseudo-stationary phases or carriers in vesicle or liposome electrokinetic chromatography (EKC) in the way as microemulsions and micelles are used; it includes the chromatographic use of liposomal bilayers as stationary phases attached to the wall for capillary electrochromatography (CEC). The second topic is the characterization and separation of liposomes as analytes by capillary electrophoresis (CE). Then the determination of distribution coefficients and binding constants between liposomes and ligands is discussed, and finally work dealing with peptides and proteins are reviewed with lipid bilayers as constituents of the electrically driven separation system.