A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the a2-macroglobulin receptor/LDLRrelated protein (a2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptorassociated protein (RAP), providing evidence that a2MR/LRP also acts as a minor group HRV receptor.Common colds most frequently arise through infection with human rhinoviruses (HRVs). The 102 antigenically distinct serotypes are divided into two groups based on receptor specificity (1, 2). The major group binds to the intercellular adhesion molecule 1 (ICAM-1) (3)(4)(5), and the minor group has been shown to attach to a membrane protein with a relative molecular mass of about 120 kDa (6, 7). ICAM-1 and the poliovirus receptor (8) are members of the immunoglobulin superfamily. As the three-dimensional structures of representative HRVs from the two different receptor groups (9, 10) and of poliovirus (11) show considerable similarity, it might have been expected that the minor group receptor would also belong to this family. However, in this communication we present evidence that minor-group HRVs gain access to the cell via members of the low density lipoprotein (LDL) receptor (LDLR) family (12,13). MATERIALS AND METHODSPurification of HRV2-Binding Protein. Two hundred liters of HeLa cell culture supernatant were concentrated ten times by ultrafiltration, dialyzed against 250 liters of H20 containing 0.02% NaN3, and adjusted to contain 20 mM N-methylpiperazine hydrochloride (pH 4.5). Precipitated material was removed, and the filtered supernatant was applied to a 0.5-liter Macroprep 50 Q column (Bio-Rad). Bound material was eluted with the same buffer containing 0.5 M NaCl. After adjustment to pH 7.2 with 1 M Tris HCl (pH 8), the material was loaded onto a 100-ml Lens culinaris lectin column (Pharmacia), and bound protein was eluted with phosphatebuffered saline (PBS) containing 0.5 M a-D-methyl glucopyranoside and precipitated with (NH4)2SO4 at 50o saturation. The precipitate was dissolved in 200 ml of PBS, the solution was passed over a 40-ml Jacalin agarose column (Vector Laboratories), and bound protein was eluted with 120 ml of 0.1 M a-D-methyl galactopyranoside in PBS and precipitated with (NH4)2SO4 as above. The precipitate was dissolved in 20 mM N-methylpiperazine hydrochloride (pH 4.5) and desalted on a PD-10 column (Pharmacia). Protein was applied onto a Mono Q HR 5/5 column (Pharmacia) and eluted with a gradient of 0-0.5 M NaCl in the same buffer. The binding activity was monitored throughout the purification procedure on ligand blots (7). Active fractions were concentrated to 1.5 ml with a Centricon-30...

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 alpha-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 (r(2) = 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.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2AP" and 3CPm. The cleavage of VPO (giving rise to VP2 and VP4). which is carried out by a so-far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases.A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dkfservices/NetPicoRNA.Keywords: cleavage site prediction; neural networks; picornavirus; proteinase; surface exposureMembers of the picornavirus family express their genomic RNA as a single polyprotein that is proteolytically processed to the mature polypeptides. At least three proteinases are required for the individual protein components to be released (reviewed in Krausslich Hellen et al., 1989;Lawson & Semler, 1990). The primary cleavage, which severs the capsid precursor PI from the nonstructural region P2-P3, is performed cotranslationally by the viral proteinase 2APm in enteroviruses and human rhinoviruses (HRVs; see Fig. I). Most of the remaining cleavages are catalyzed by the viral proteinase, 3CP". In cardio-, hepato-, and aphthoviruses, which also belong to the picornavirus family, the L-proteinase performs functions similar to those of 2APm, resulting in a somewhat different cleavage scheme (see Fig. I). Concomitantly with RNA encapsidation, VPO is cleaved to VP4 and VP2; it is believed that the RNA itself exerts a catalytic function in this event (Arnold et a!., 1987;Harber et al., 1991;Bishop &Anderson, 1993;Basavappa et al., 1994).In addition to processing of the viral polyprotein, the proteinases also cleave cellular targets. When infected with poliovirus, at least nine acidic and five basic cellular proteins were shown to be degraded in two-dimensional gel electrophoresis (Urzanqui & Carrasco, 1989). The degradation of cellular proteins seems to be part of the viral attack mechanism, leading to host cell shur-ofJ-a decrease in cellular transcription and translation that has no influence on viral replication. The best-studied event is the cleavage of the eukaryotic initiation factor 4G (eIF-4G). which is required for cap-dependent translation of cellular mRNA. This protein is degraded by 2APm in entero-and...

No abstract

A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA. Biochemical and genetic evidence suggests that the viral proteinase 2A induces cleavage of the eukaryotic initiation factor (eIF) 4 gamma (also known as p220) component of eIF-4 (formerly called eIF-4F). However, neither the mechanism underlying the specific proteolysis of eIF-4 gamma nor the influence of this cleavage on the translation of capped mRNAs has been clarified. Such studies have been hampered by a lack of large quantities of a purified 2A proteinase. Therefore, the mature proteinases 2A of human rhinovirus 2 and coxsackievirus B4 were expressed in soluble form in Escherichia coli. A four-step purification protocol was developed; 1 mg of highly purified 2A proteinase per gram wet weight of E. coli was obtained. Both enzymes cleaved directly eIF-4 gamma as part of the purified eIF-4 complex. Addition of HRV2 2A proteinase to HeLa cell cytoplasmic translation extracts resulted in eIF-4 gamma cleavage and drastically reduced the translation of capped mRNA; addition of purified eIF-4 restored translation to the initial level. However, translation of a reporter gene driven by the 5'-untranslated region of human rhinovirus 2 was translated 2-3-fold more efficiently in the presence of HRV2 2A proteinase.

Minor group human rhinoviruses (HRVs) attach to members of the low-density lipoprotein receptor family and are internalized via receptor-mediated endocytosis. The attachment of HRV2 to the cell surface, the first step in infection, was characterized at the singlemolecule level by atomic force spectroscopy. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra, which suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several hundred milliseconds. The number of receptors involved in virus binding was determined, and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane-anchored receptors were obtained.force spectroscopy ͉ molecular recognition ͉ single virus binding ͉ very low density lipoprotein receptor ͉ picornavirus H uman rhinoviruses (HRVs), members of the Picornaviridae family, are the most frequent cause of colds. Their icosahedral capsid (30 nm in diameter) is built from 60 copies each of 4 viral proteins VP1, VP2, VP3, and VP4 that surround the RNA genome. Of the 99 so far characterized serotypes, 12 (the minor receptor group) bind low-density lipoprotein receptor (LDLR), very-LDLR (VLDLR), and LDLR-related protein (LRP) (1, 2). This receptor family functions in endocytosis and signal transduction recognizing a variety of ligands (3). LDLR and VLDLR possess 5 domains (4), including an N-terminal ligand-binding domain composed of 7 (LDLR; L1-L7) and 8 (VLDLR; V1-V8) modules, a region similar to the EGFprecursor and a ␤-propeller with YWTD motifs that is implicated in low pH-induced release of the ligands in endosomes (5). Adjacent to the plasma membrane is a domain carrying O-linked oligosaccharides followed by the transmembrane anchor and the carboxyl terminus carrying a NPXY clathrin localization signal. The ligand binding modules are Ϸ40 amino acid residues in length. They are stabilized by a Ca ion and 6 highly conserved cysteines forming disulfide bridges (6). Differences in the types and numbers of repeats allow for recognition of a large variety of structurally and functionally diverse ligands.For infection, HRV2 attaches to LDLR and/or LRP at the cell membrane. It can be released with EDTA immediately after attachment to the cell but within some minutes becomes tightly bound and not dissociable (7). This finding was taken to indicate either recruitment of multiple receptors, thus enforcing an initial bond with a single receptor, and/or engulfment within membranes as the virus enters in clathrin coated vesicles (8). Subsequently, it presumably dissociates from its receptors upon arrival in the mildly acidic milieu (pH 6.5-6.0) of early endosomes (9); finally, the virus is delivered to endosomal carrier vesicles and late endosomes from where its RNA genome is released into the cytosol.Performing single-molecule force spectroscopy with an atomic force microscop...

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.