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Interaction of poliovirus with its purified receptor and conformational alteration in the virion
Abstract:
Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 +/- (0.86) x 10(-8) M at 4 degrees C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37 degrees C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.
Insights
Human poliovirus receptor (hPVR) fragments PVR241 and PVR330 were produced and purified. PVR330 binds poliovirus virions, causing conformational changes and suggesting a specific interaction mechanism.
Area of Science:
- Biochemistry
- Virology
- Molecular Biology
Background:
- The human poliovirus receptor (hPVR) facilitates poliovirus entry into host cells.
- Understanding hPVR-poliovirus interactions is crucial for antiviral strategies.
- Extracellular domains of hPVR play a key role in virus binding.
Purpose of the Study:
- To produce and characterize functional fragments of the human poliovirus receptor (hPVR).
- To investigate the binding kinetics and conformational effects of hPVR-poliovirus interactions.
- To elucidate the mechanism of poliovirus conformational changes upon receptor binding.
Main Methods:
- Production of hPVR polypeptides (PVR241, PVR330) using a baculovirus expression system.
- Purification of hPVR fragments via immunoaffinity chromatography with anti-hPVR monoclonal antibodies.
- In vitro binding assays mixing purified PVR with poliovirus type 1 (Mahoney strain).
- Analysis of virus conformational changes (160S to 135S and 80S particles) using sedimentation assays.
Main Results:
- Purified PVR241 and PVR330 retained native conformation, confirmed by antibody reactivity.
- PVR330 demonstrated binding to poliovirus virions, with at least 43 PVR330 molecules binding per virion.
- The equilibrium dissociation constant (Kd) for PVR330-virion interaction was determined as 4.50 x 10(-8) M at 4°C.
- Increased PVR330 concentration led to higher rates of poliovirus conformational changes (160S to 135S and 80S particles).
- The ratio of 135S to 80S particles remained constant, and 80S particles did not form from 135S particles post-160S disappearance.
Conclusions:
- Recombinant hPVR fragments, particularly PVR330, can be produced and retain functional conformation.
- PVR330 binds poliovirus virions with high affinity, indicating specific molecular interactions.
- PVR binding induces significant conformational changes in the poliovirus capsid, potentially related to uncoating or entry mechanisms.
- The observed conformational transitions suggest distinct pathways or intermediates in the virus-receptor interaction process.