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Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation
H Kowalski1, I Maurer-Fogy, G Vriend
1Institut für Biochemie, University of Vienna, Wien, Austria.
Abstract:
Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.
Insights
Human rhinovirus (HRV) serotypes show varying sensitivity to trypsin. Low pH treatment exposes cleavage sites on HRV capsids, enabling trypsin degradation of viral proteins.
Area of Science:
- Virology
- Structural Biology
- Biochemistry
Background:
- Human rhinoviruses (HRVs) are common respiratory pathogens.
- Understanding HRV capsid stability is crucial for antiviral development.
- Serotype-specific differences in HRV protein stability are not fully understood.
Purpose of the Study:
- To investigate the trypsin sensitivity of different human rhinovirus (HRV) serotypes.
- To determine the effect of low pH treatment on HRV capsid structure and protein accessibility.
- To identify specific cleavage sites within HRV structural proteins.
Main Methods:
- Enzymatic assays using trypsin on five HRV serotypes.
- Low pH treatment followed by neutralization to induce conformational changes.
- Direct protein sequencing to identify trypsin cleavage sites.
- Analysis of three-dimensional structural maps of HRV2.
Main Results:
- HRV1A, HRV2, and HRV14 were resistant to trypsin at physiological pH, while HRV49 and HRV89 showed VP2 degradation.
- Low pH treatment exposed VP1 cleavage sites in HRV1A, HRV2, and HRV49, followed by VP2 degradation.
- The VP1 cleavage site in HRV2 was identified between Arg260 and Thr261.
- HRV14 and HRV89 showed resistance to cleavage even after low pH treatment.
- The cleavage site in native HRV2 is structurally inaccessible, indicating conformational changes are required.
Conclusions:
- HRV capsid conformation dictates trypsin accessibility and cleavage susceptibility.
- Low pH-induced structural rearrangements are necessary for trypsin-mediated VP1 and VP2 degradation.
- Specific amino acid sequences and structural accessibility influence HRV protein cleavage by trypsin.