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The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

Rhinoviruses (RVs) and coronaviruses (CoVs) upregulate host cell metabolic pathways such as glycolysis to meet their bioenergetic demands for rapid multiplication. Using the glycolysis inhibitor 2-deoxy-D-glucose (2-DG), we assessed the dose-dependent inhibition of viral replication of minor- and major-receptor group RVs in epithelial cells. 2-DG disrupted RV infection cycle by inhibiting template negative-strand as well as genomic positive-strand RNA synthesis, resulting in less progeny virus and RV-mediated cell death. Assessment of 2-DG´s intracellular kinetics revealed that after a short-exposure to 2-DG, the active intermediate, 2-DG6P, is stored intracellularly for several hours. Finally, we confirmed the antiviral effect of 2-DG on pandemic SARS-CoV-2 and showed for the first time that 2-DG also reduces replication of endemic human coronaviruses (HCoVs). These results provide further evidence that 2-DG could be utilized as a broad-spectrum antiviral.

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 detectedviatheir 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.

ABSTRACTHuman rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.

ABSTRACTMajor receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a “catalytic” function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the ‘cone jet’ mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an α-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r² = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of ±5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided. Copyright © 2001 John Wiley & Sons, Ltd.

We used dynamic force microscopy to study the attachment of human rhinovirus 2 (HRV2) particles to model membranes. A supported lipid bilayer containing Ni²⁺-nitrilotriacetate (NTA)-lipids as functional groups in the outer leaflet at high surface density was assembled on mica and showed a flat extended structure of 3 nm in height. The recombinant very-low density lipoprotein receptor fragment MBP-VLDLR1-8, consisting of the ligand binding domain (all eight complement type A repeats) fused at the N-terminus to maltose binding protein (MBP) and containing a hexahistidine (His6)-tag at its C-terminus, was attached to the membranes via the His6-Ni²⁺NTA bond and revealed monolayer density. The protein-lipid layer was then used as a model to investigate virus binding to its receptor in a planar membrane. The viral particles attached in a tightly packed, long range order quasi-crystalline arrangement. This assembly is thus ideally suited for studies of viral dynamics and recognition under physiological conditions.

Minor group human rhinoviruses (exemplified by human rhinovirus serotype 2 (HRV2)) use members of the low density lipoprotein receptor family for cell entry; all these receptors possess clathrin-coated pit localization signals. Viral infection should thus be inhibited under conditions of impaired clathrin-mediated endocytosis. However, Madshus et al.reported an increase in the cytopathic effect of HRV2 infection in HEp-2 cells upon suppression of clathrin-dependent endocytosis by hypotonic shock and potassium depletion (Madshus, I. H., Sandvig, K., Olsnes, S., and van Deurs, B. (1987) J. Cell. Physiol.131, 14–22.) To resolve this apparent contradiction, we investigated the binding, internalization, conformational changes, and productive uncoating of HRV2 in HeLa cells subjected to hypotonic shock and potassium depletion. This treatment led to an increase in HRV2 binding, with internalization being barely affected. The generation of C-antigenic particles requiring pH ≤5.6 was strongly reduced due to an elevation of the pH in endosomal compartments. However, K+depletion only slightly affected de novoviral protein synthesis, suggesting that productivity of viral RNA in the cytoplasm is enhanced and thus compensates for the reduction in C-antigenic particles. The distinct steps in the entry pathway of HRV2 are thus differently influenced by potassium depletion. Viral internalization under conditions of inhibited clathrin-dependent endocytosis without the need to disturb the ionic milieu was confirmed in HeLa cells overexpressing the nonfunctional dynamin-1 mutant K44A. Unexpectedly, overexpression of dynamin-1 K44A resulted in elevated endosomal pH compared with overexpression of wild-type dynamin.

The formation of complexes between the minor receptor group human rhinovirus HRV2 and two recombinant soluble receptor fragments derived from the human very low density lipoprotein receptor (VLDLR) and containing ligand-binding repeats 1–3 (MBP·VLDLR1–3) or 1–8 (MBP·VLDLR1–8) fused to the carboxyl terminus of the maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the determination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versustotal virus concentration. Extrapolation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components present in the complex. Using this method, we determined that about 60 molecules of MBP·VLDLR1–3but only about 30 molecules of MBP·VLDLR1–8were bound per virion.

Minor group human rhinoviruses (HRVs) use members of the low-density lipoprotein receptor family for cell entry. To investigate the utility of receptor fragments as viral inhibitors, various polypeptide segments derived from the ligand binding domain of human very-low-density lipoprotein receptor (VLDLR) were expressed in a soluble form in bacteria. Whereas none of the fragments was active in virus binding immediately after recovery from the cell lysates, constructs encompassing complement type repeats 1-3, 1-6, and 1-8 spontaneously acquired virus binding activity by incubation at 4°C in buffer containing Ca2+ions and lacking any redox system. When immobilized receptor-associated protein (RAP), a specific chaperone for VLDLR, was present during the incubation, the yield of protein active in ligand binding was substantially increased. A VLDLR fragment with repeats 4–6 failed to bind virus; however, it bound RAP. Bacterial expression of truncated VLDLR 1-3 at high yield, easy purification, and folding together with high inhibitory activity toward HRV2 makes this protein a promising starting point for the development of an oligopeptide-based antiviral agent. Using sucrose density gradient centrifugation, we demonstrate the formation of virus–receptor complexes. The recombinant receptors can thus be used for structure determination by electron cryo-microscopy.

Determination of infectious progeny virus and in vivo labelling with [35S]methionine followed by immunoprecipitation demonstrates that the major receptor group human rhinovirus HRV14 is able to infect HeLa cells in the presence of the V‐ATPase inhibitor bafilomycin A1. However, host cell shut off is delayed and viral yield is decreased in the presence of the drug. Uncoating can thus take place under conditions that prevent endosomal acidification indicating that it is catalysed by the viral receptor alone. Since transport is arrested in early endosomes upon inhibition of vesicle acidification, the data also suggest that productive uncoating takes place from early endocytic compartments.

Screening of a phage library displaying single chain fragments of the variable regions of human immunoglobulins (scFv) for binding to the ovarian chicken very low-density lipoprotein/vitellogenin receptor (OVR) led to the isolation of several antibody fragments with high affinity. As for the natural ligands of OVR, receptor binding of all antibody fragments is strictly Ca 2+-dependent and is prevented by receptor-associated protein (RAP). Moreover, attachment of human rhinovirus serotype 2 (HRV2) to this receptor is inhibited by all scFvs. In contrast to conventional immunization, the in vitro selection method thus exclusively led to antibodies that attach to or close to the ligand binding site and thereby block the receptor-ligand interaction.

ABSTRACTThe large family of human rhinoviruses, the main causative agents of the common cold, is divided into the major and the minor group based on receptor specificity. Major group viruses attach to intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, whereas minor group viruses use low-density lipoprotein receptors (LDLR) for cell entry. During early attempts aimed at isolating the minor group receptor, we discovered that a protein with virus binding activity was released from HeLa cells upon incubation with buffer at 37°C (F. Hofer, B. Berger, M. Gruenberger, H. Machat, R. Dernick, U. Tessmer, E. Kuechler, and D. Blaas, J. Gen. Virol. 73:627–632, 1992). In light of the recent discovery of several new members of the LDLR family, we reinvestigated the nature of this protein and present evidence for its being derived from the human very-low density lipoprotein receptor (VLDLR). A soluble VLDLR fragment encompassing the eight complement type repeats and representing the N-terminal part of the receptor was then expressed in the baculovirus system; both the shed protein and the recombinant soluble VLDLR bind minor group viruses and inhibit viral infection of HeLa cells in a concentration-dependent manner.

ABSTRACTBafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

ABSTRACTIntercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111–123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff= 2 × 108M−1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/α2MR), and it competes for binding of receptor-associated protein to LRP/α2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/α2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.

A fragment of the low density lipoprotein receptor encompassing the seven ligand binding repeats was expressed in Sf9 insect cells as a fusion protein with a carboxyl‐terminally linked hexa‐his tag by using a baculovirus vector. Up to 10 mg/l of the fusion protein was secreted into the medium. The material was soluble in the absence of detergent and active in binding β very low density lipoprotein and a member of the minor group of human rhinoviruses (HRV2) in ligand blots from sodium dodecyl sulfate‐polyacrylamide gels run under nonreducing conditions. The receptor fragment specifically inhibits viral infection of HeLa cells by minor group HRVs in a concentration‐dependent manner. Viral infectivity is neutralized by aggregation.—Marlovits, T. C., Zechmeister, T., Gruenberger, M., Ronacher, B., Schwihla, H., Blaas, D. Recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro. FASEB J.12, 695–703 (1998)

Screening of a phage library displaying single chain fragments of the variable regions of human immunoglobulins (scFv) for binding to the ovarian chicken very low‐density lipoprotein/vitellogenin receptor (OVR) led to the isolation of several antibody fragments with high affinity. As for the natural ligands of OVR, receptor binding of all antibody fragments is strictly Ca2+‐dependent and is prevented by receptor‐associated protein (RAP). Moreover, attachment of human rhinovirus serotype 2 (HRV2) to this receptor is inhibited by all scFvs. In contrast to conventional immunization, the in vitro selection method thus exclusively led to antibodies that attach to or close to the ligand binding site and thereby block the receptor‐ligand interaction.

ABSTRACTThe structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-Å resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45° to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2–Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell‐attachment site; ten serotypes (the minor group) bind to members of the low‐density lipoprotein receptor. Three different crystal forms of the minor‐group human rhinovirus serotype 2 (HRV2) were obtained by the hanging‐drop vapour‐diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P21, diffracted at least to 2.8 Å resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 Å resolution. These crystals belong to a primitive orthorhombic space group, with unit‐cell parameters a= 309.3, b= 353.5, c= 759.6 Å, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit‐cell parameters a= 308.7, b= 352.2, c= 380.5 Å, diffracted to high resolution (beyond 1.8 Å) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

A fragment of the human low‐density lipoprotein receptor encompassing the seven ligand‐binding repeats fused to a C‐terminal oligo‐His tag was expressed in Sf9 insect cells. The melittin signal sequence encoded in the baculovirus vector led to secretion of the protein into the cell supernatant in a soluble form. The receptor fragment bound its natural ligand β‐migrating very‐low‐density lipoprotein and human rhinovirus serotype 2 in non‐reducing ligand blots. Infection of all minor group human rhinovirus serotypes investigated was inhibited by the presence of the receptor fragment during viral challenge of HeLa cells. Infection is inhibited by aggregation of the virions. Copyright © 1998 John Wiley & Sons, Ltd.

A fragment of the human low-density lipoprotein receptor encompassing the seven ligand-binding repeats fused to a C-terminal oligo-His tag was expressed in Sf9 insect cells. The melittin signal sequence encoded in the baculovirus vector led to secretion of the protein into the cell supernatant in a soluble form. The receptor fragment bound its natural ligand β-migrating very-low-density lipoprotein and human rhinovirus serotype 2 in non-reducing ligand blots. Infection of all minor group human rhinovirus serotypes investigated was inhibited by the presence of the receptor fragment during viral challenge of HeLa cells. Infection is inhibited by aggregation of the virions. Copyright © 1998 John Wiley & Sons, Ltd.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5′end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3′ half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5′ half (including the capsid proteins) are more heterogeneous.

A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.