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Cleavage of viral precursor proteins in vivo and in vitro
Abstract:
The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.
Insights
Protease inhibitors prevent viral polyprotein cleavage in infected cells, with effectiveness varying by cell type. Host cell proteases contribute to polyprotein processing, suggesting complex viral replication mechanisms.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Viral replication involves the synthesis of large precursor polyproteins.
- Proteolytic processing is essential for generating functional viral proteins.
- Host cell machinery can play a role in viral protein maturation.
Purpose of the Study:
- To investigate the effect of protease inhibitors on viral polyprotein accumulation.
- To determine the role of host cell proteases in viral polyprotein cleavage.
- To characterize the in vitro cleavage products of viral polyprotein.
Main Methods:
- Infection of tissue culture cells (monkey kidney, HeLa) with poliovirus or echovirus 12.
- Treatment of infected cells with specific protease inhibitors (chymotrypsin, trypsin).
- Analysis of polyprotein cleavage using SDS-PAGE and antigenicity assays.
- In vitro cleavage assays with extracted viral polyprotein and cell extracts.
Main Results:
- Protease inhibitors caused accumulation of large viral polyproteins in infected cells.
- Inhibitor effectiveness was cell-line dependent, indicating host cell protease involvement.
- Chymotrypsin cleavage of poliovirus polyprotein in vitro produced fragments similar to in vivo products.
- Viral polyprotein was associated with a membrane-bound structure, not soluble.
- Cell extracts from infected and uninfected cells showed distinct proteolytic activities.
Conclusions:
- Host cell proteases contribute to viral polyprotein processing.
- Viral polyprotein cleavage is influenced by both viral and host factors.
- Understanding these mechanisms is crucial for developing antiviral strategies.