At the concentration of sPvr used, 1. These results indicate that sPvr produced in mammalian cells can efficiently bind to poliovirus and induce the structural changes associated with cell entry. Kinetics of sPvr-induced conformational changes of poliovirus. Surface plasmon resonance allows determination of quantitative affinities K D , association k a , and dissociation k d rates for the formation and dissociation of the virus-receptor complex To examine the kinetics of binding of sPvr to poliovirus by surface plasmon resonance, purified poliovirus was coupled to the sensor chip surface, and sPvr was injected over the chip surface.
An example of raw sensorgram data is shown in Fig. In this experiment, flow cell 2 contained immobilized poliovirus, and flow cell 1 was activated and blocked without virus. At s, sPvr was replaced with buffer, and the dissociation of complex was followed for 3 min. The response on the y axis is measured in response units.
The sensorgram reveals a change in the bulk refractive index, but there was no significant background response when 1. In the surface plasmon resonance experiments that followed, data from flow cell 2 were subtracted from the data from flow cell 1 to correct for changes in bulk refractive index.
These results demonstrate binding of sPvr to poliovirus immobilized on the chip surface. Example of raw sensorgram data.
At s, the sample was replaced with buffer, and dissociation was followed for 2 min. The sensor chip surface was regenerated by treatment with low pH, to disrupt the virus-receptor interaction. Poliovirus remaining on the chip surface should survive these conditions, because its natural route of infection is through the acidic environment of the stomach. Two experiments were done to ensure that the sensor chips could be reused. First, unbound poliovirus was incubated in regeneration buffer glycine buffer, pH 3 for 5 min at room temperature, and then infectivity was determined by plaque assay.
As expected, this treatment did not reduce poliovirus infectivity, suggesting that conditions used for regeneration of the sensor chip do not disrupt virus structure Fig. Second, repeated use and regeneration of sensor chips containing bound poliovirus did not affect sensorgrams and response levels data not shown. Effect of low pH treatment on poliovirus infectivity.
Approximately 70 pfu of poliovirus were incubated in 10 m m glycine, pH 3, or PBS, for 5 min, followed by plaque assay. Shown is the average of two experiments. To determine the specificity of the poliovirus-sPvr interaction, a blocking experiment was performed using a monoclonal antibody, C, directed against the first domain of Pvr and which prevents poliovirus attachment to cells Preincubation with monoclonal antibody C inhibited formation of the virus-receptor complex Fig.
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A control monoclonal antibody DL11, directed against herpes simplex virus glycoprotein D, did not inhibit the formation of the poliovirus-sPvr complex data not shown. These results indicate that the sPvr-poliovirus interaction under study resembles the interaction during infection of cells, since it is mediated by domain 1 of sPvr. Specificity of sPvr interaction with poliovirus on sensor chip surface.
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At s, the sample was replaced with buffer, and dissociation followed for 3 min. The sensorgrams of the sPvr-poliovirus interaction were imposed upon different model curves generated by global fitting analysis Fig. On the other hand, the X 2 value for a one-site binding model was 29, demonstrating a poor fit to that model. The two affinity constants calculated from the surface plasmon resonance data are 0.
The calculated association rate constants are 3. Binding rates were unaffected by changes in flow rate, demonstrating that the poliovirus-sPvr interaction is not limited by mass transport data not shown The kinetics and affinity analysis of the rhinovirus-sICAM interaction using the biosensor, as well as affinity analysis in solution, was also shown to be biphasic In that study, the linear transformation method was used to analyze biosensor data on the rhinovirus-sICAM-1 interaction. This method, when applied to our data on the poliovirus-sPvr interaction, also yields biphasic plots indicative of two binding sites data not shown.
Corrected sensorgram overlays for the interaction of decreasing concentrations of sPvr with immobilized poliovirus. Data were collected at 5 Hz. The black lines are the best global fit to the parallel reactions model BIAevaluation 3. Binding of sPvr to the sensor chip was repeated under equilibrium conditions to confirm the existence of two classes of binding sites, and the affinity constants were determined by Scatchard analysis The contact time was varied from 50 min for the lowest concentration to 10 min for the highest concentration of sPvr Fig.
These values are similar to those obtained by kinetic analysis Table II. Equilibrium binding sensorgrams and Scatchard analysis of the binding of sPvr to immobilized poliovirus.
A, binding of sPvr to immobilized poliovirus was monitored for 10 min for the injections of Arrows indicate the time points used for the Scatchard analysis. B, Scatchard analysis. The negative slope of each line is equal to each association constant; the reciprocals are the K D values. The R 2 values for the linear fit of the data were 0. Higher temperatures, at which receptor-induced virus disruption occurs, were not studied because it would be difficult to interpret the biosensor data Binding of sPvr at these sites on poliovirus is therefore endothermic. The value for K D 2 did not exhibit a general increase or decrease with temperature, and therefore the thermodynamic nature of this site could not be determined.
Affinity constants for sPvr binding to poliovirus type 1 and abundance of each binding class at different temperatures. The relative abundance of the K D 1 and K D 2 sites at different temperatures was calculated from the kinetics data using global analysis software, assuming a parallel reactions model. The relative abundance of the K D 2 site decreased with decreasing temperature. To measure kinetic constants of the poliovirus-receptor interaction, we expressed and purified from mammalian cells a soluble form of the poliovirus receptor.
Surface plasmon resonance was used to study binding of poliovirus with sPvr. The affinities determined by biosensor are within 1 order of magnitude of the IC 50 of sPvr determined by plaque assay, suggesting that the values determined by BIAcore could be the functional affinities for sPvr.
The results indicate that the interaction between poliovirus and sPvr is biphasic. Two classes of binding site for sPvr on poliovirus were detected, called the K D 1 site and the K D 2 site. The fraction of K D 2 sites, with a binding affinity of 0. A biphasic binding model for poliovirus and Pvr has not been described previously. We find that the binding affinity of the K D 1 site, the predominant binding site at this temperature, is 4 orders of magnitude lower.
The difference may be explained by the fact that the binding affinities calculated in the present study represent the intrinsic affinity of poliovirus for a single receptor molecule. In contrast, receptor molecules may cluster on the cell surface, increasing the apparent affinity, or avidity, of the virus-receptor interaction.
Such clustering does not occur in solution In another study, a single binding affinity of poliovirus for a soluble form of Pvr produced in insect cells was determined to be 4. In those studies, binding assays were conducted in plastic microtiter plates. Although the affinity of this site is similar to that of the K D 2 site, it is not clear why the lower affinity site was not detected.
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One possibility is that concentrations of sPvr were not sufficiently high to detect the lower affinity site. In addition, proteins produced in insect cells and in mammalian cells have different patterns of glycosylation, which might contribute to the different results. An N -linked glycosylation site within Pvr domain 1 is known to influence its interactions with poliovirus 12 and may contact the receptor binding site on the viral capsid 6.
A side-by-side comparison must be done to resolve this issue. The finding of two classes of receptor-binding sites on a virus has also been reported for rhinovirus type 3 and a soluble form of its cellular receptor, ICAM-1 38 , Although the rhinovirus-sICAM and poliovirus-sPvr interactions are biphasic, there are significant differences in the affinity and kinetic constants. The greater association rate of poliovirus-sPvr might be due, in part, to differences in the extent of contact between virus and receptor. Three-dimensional models of virus-receptor complexes produced from cryo-electron microscopy and image reconstruction reveal that the footprint of Pvr on poliovirus is significantly larger than that of ICAM-1 on rhinovirus 6 , 7 , The extra surface area on poliovirus includes the knob of VP3 and the C terminus of VP1 from the 5-fold related promoter in the southeast corner of the road map describing the contact of Pvr on poliovirus 6.
In contrast, although there are two dissociation rate constants for poliovirus-sPvr, only one has been reported for the rhinovirus 3-sICAM interaction 38 , The dissociation rates for the poliovirus-sPvr interaction are 1. Consistent with these differences is the fact that the IC 50 of sICAM-1 for rhinovirus 3 is fold higher than that of poliovirus However, other factors might play a role, including the number of receptors per virus particle that are required to neutralize infectivity.
The effect of temperature on the interaction of poliovirus with sPvr was studied. Binding at the lower affinity site, K D 1 , is endothermic e. As suggested previously, heat absorbed during the interaction of virus with receptor might help to lower the energy barrier required for uncoating of the virus particle The affinity of this interaction is at least 4 times lower than either of the binding sites on poliovirus for sPvr.
Like most protein-protein interactions, the affinity of echovirus 11 for CD55 increases with decreased temperature, indicating that binding is exothermic. The association rate for the interaction between echovirus 11 and CD55 is faster than that of poliovirus-sPvr and 4. One explanation for these findings is that the contact between echovirus 11 and CD55 is more extensive than that of the other two virus-receptor complexes. In addition, the binding site for CD55 on echovirus 11 might be more accessible than those of Pvr and ICAM-1, which are located in a depression on the capsid 6 , 7.
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The dissociation rate for the echovirus-CD55 interaction is at least 97 times faster than that of either poliovirus-sPvr or rhinovirus-sICAM-1 These findings are consistent with a more accessible binding site for CD55 on echovirus 11, compared with the receptor-binding sites on poliovirus and rhinovirus 38 , In addition, it is possible that the atomic interactions between CD55 and echovirus 11 are weaker than between the other two viruses and their receptors. The lower dissociation rates for the poliovirus- and rhinovirus-receptor complexes may in part reflect the time required for structural changes to occur.
Elucidation of the high resolution crystal structures of all three virus-receptor complexes should provide explanations for the differences in kinetic parameters. Why do poliovirus and rhinovirus have two classes of receptor-binding sites? We do not guarantee individual replies due to extremely high volume of correspondence. E-mail the story Virus may jump species through 'rock-and-roll' motion with receptors Your friend's email Your email I would like to subscribe to Science X Newsletter.
Researchers report the rock-and-roll motion of the canine parvovirus when it comes in contact with the transferrin receptor receptor offers hints at how the disease was able to evolve and strike several different species.
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Sep 23, How have scientists derived the gene sequences known today? Related Stories. First accurate simulation of a virus invading a cell Sep 12, Jan 24, In viruses with a viral envelope , viral receptors attach to the receptors on the surface of the cell and secondary receptors may be present to initiate the puncture of the membrane or fusion with the host cell. Following attachment, the viral envelope fuses with the host cell membrane, emptying the now-bare virus into the cell.
In essence, the virus's envelope "blends" with the host cell membrane, releasing its contents into the cell. Obviously, this can only be done with viruses that have an envelope examples of such enveloped viruses include HIV , KSHV     and herpes simplex virus. Viruses with no viral envelope enter the cell through endocytosis ; they are ingested by the host cell through the cell membrane. In essence, the virus tricks the cell into thinking that the virus knocking at the door is nothing more than nutrition or harmless goods. A cell, which naturally takes in resources from the environment by attaching goods onto surface receptors and bringing them into the cell, will engulf the virus.
Once inside the cell, the virus must now break out of the vesicle by which it was taken up in order to gain access to the cytoplasm. Examples include the poliovirus , Hepatitis C virus  and Foot-and-mouth disease virus. Many enveloped viruses also enter the cell through endocytosis. Entry via the endosome guarantees low pH and exposure to proteases which are needed to open the viral capsid and release the genetic material inside. Further, endosomes transport the virus through the cell and ensure that no trace of the virus is left on the surface, which could be a substrate for immune recognition.
A third and more specific example, is by simply attaching to the surface of the cell via receptors on the cell, and injecting only its genome into the cell, leaving the rest of the virus on the surface. This is restricted to viruses in which only the gene is required for infection of a cell most positive-sense, single-stranded RNA viruses because they can be immediately translated and further restricted to viruses that actually exhibit this behavior.
The best studied example includes the bacteriophages ; for example, when the tail fibers of the T2 phage land on a cell, its central sheath pierces the cell membrane and the phage injects DNA from the head capsid directly into the cell. Once a virus is in a cell, it will activate formation of proteins either by itself or using the host to gain full control of the host cell, if it is able to. Control mechanisms include the suppression of intrinsic cell defenses, suppression of cell signaling and suppression of host cellular transcription and translation.
Often, it is these cytotoxic effects that lead to the death and decline of a cell infected by a virus. A cell is classified as susceptible to a virus if the virus is able to enter the cell. After the introduction of the viral particle, unpacking of the contents viral proteins in the tegument and the viral genome via some form of nucleic acid occurs as preparation of the next stage of viral infection: viral replication.
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