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Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin
Abstract:
Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatment of infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R x K/R R. Uncleaved gB precursor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.
Insights
This study shows that inhibiting specific cellular proteases prevents human cytomegalovirus glycoprotein B (gB) cleavage. This inhibition allows gB precursor accumulation, indicating normal cellular transport despite drug treatment.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Human cytomegalovirus (HCMV) glycoprotein B (gB) undergoes endoproteolytic cleavage for its maturation and function.
- Cellular subtilisin-like endoproteases are implicated in this critical processing step.
- Understanding gB processing is vital for developing antiviral strategies.
Purpose of the Study:
- To investigate the role of cellular subtilisin-like endoproteases in HCMV gB cleavage.
- To determine if inhibiting these proteases affects gB maturation and transport.
- To identify specific proteases involved in gB processing.
Main Methods:
- Treatment of HCMV-infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), a protease inhibitor targeting the R-X-K/R-R motif.
- Analysis of uncleaved gB precursor molecules using endoglycosidase H resistance assays.
- Infection of CV-1 cells with recombinant vaccinia virus expressing gB (VVgB).
- Coinfection experiments with VVgB and a recombinant vaccinia-human furin construct.
Main Results:
- decRVKR-CMK treatment inhibited endoproteolytic cleavage of HCMV gB.
- Accumulated uncleaved gB precursor molecules (160 kDa) were endoglycosidase H resistant, suggesting proper cellular transport.
- The inhibitor prevented gB processing in VVgB-infected cells.
- Coinfection with VVgB and a furin construct restored gB endoproteolytic activity, implicating furin.
Conclusions:
- Cellular subtilisin-like endoproteases, particularly furin, are directly involved in the endoproteolytic cleavage of HCMV gB.
- Inhibition of these proteases blocks gB processing but does not impede its cellular transport.
- Targeting furin or related proteases represents a potential strategy for controlling HCMV infection.