Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2

Area of Science:

• Cellular Biology
• Virology
• Structural Biology

Background:

• Tetraspanin CD81 is crucial for cellular functions and serves as an entry receptor for Hepatitis C virus (HCV).
• The interaction between CD81's large extracellular loop (LEL) and HCV glycoprotein E2 is vital for viral entry and has immunomodulatory effects.
• Understanding the structural basis of CD81 function, particularly its LEL dimerization, is key to understanding HCV entry.

Purpose of the Study:

• To investigate the relationship between the dimeric crystal structure of CD81's large extracellular loop (LEL) and the intact CD81 protein.
• To identify specific amino acids within the CD81 LEL that are critical for dimerization and their impact on E2 binding.
• To determine if mutations affecting LEL dimerization in isolation have similar effects in the context of full-length CD81.

Main Methods:

• Random mutagenesis of the CD81 LEL to identify amino acid substitutions affecting dimerization.
• Analysis of mutations impacting intermonomer contacts, salt bridge formation, and disulfide bonding within the LEL.
• Assessment of E2 binding affinity for both isolated LEL mutants and full-length CD81 mutants.
• Oligomerization studies of full-length CD81 with introduced LEL mutations.

Main Results:

• Specific mutations (F150S, V146E, K124T, T166I, C157S, C190R) were identified that abolish LEL dimerization.
• Two monomeric LEL mutants (K124T and V146E) retained the ability to bind HCV E2.
• Mutations introduced into full-length CD81 (K124T, V146E, F150S) did not disrupt oligomerization and had less severe effects on E2 binding compared to isolated LEL.
• These findings suggest that regions outside the LEL contribute to CD81 dimerization in the intact protein.

Conclusions:

• The large extracellular loop (LEL) of CD81 has a more stable structure within the intact tetraspanin compared to its isolated form.
• Amino acids outside the LEL play a significant role in the dimerization of full-length CD81.
• Understanding these structural dynamics is crucial for developing strategies to block HCV entry via CD81.