The role of the S-S bridge in retroviral protease function and virion maturation.
Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.
Zábranská
Helena
H
Gilead Sciences Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo námestí 2, 166 10 Prague 6, Czech Republic.
Tůma
Roman
R
Kluh
Ivan
I
Svatos
Ales
A
Ruml
Tomás
T
Hrabal
Richard
R
Pichová
Iva
I
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004795:Enzyme Stability; D015683:Gene Products, gag / Q000378:metabolism; D007700:Kinetics; D016093:Mason-Pfizer monkey virus / Q000201:enzymology* / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011499:Protein Processing, Post-Translational; D012192:Retroviridae Infections; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014771:Virion / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology
D004220:Disulfides; D015683:Gene Products, gag; D050505:Mutant Proteins; D010450:Endopeptidases; C445563:Mason-Pfizer monkey virus protease; D003545:Cysteine; D003488:Cyanogen Bromide