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Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy
Published on: November 4, 2018
Dissection of functional sites in herpesvirus saimiri complement control protein homolog
Malik Johid Reza1, Ashish Kamble, Muzammil Ahmad
1National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune, India.
Insights
Herpesvirus saimiri
Area of Science:
- Immunology
- Virology
Background:
- Herpesvirus saimiri encodes a complement control protein homolog (CCPH), a viral mimic of human complement regulators.
- CCPH inhibits the complement system via cofactor and decay-accelerating activities.
- Understanding CCPH's functional sites is crucial for its therapeutic potential.
Purpose of the Study:
- To fine-map the functional sites of Herpesvirus saimiri's complement control protein homolog (CCPH).
- To investigate the role of conserved residues in CCPH's cofactor and decay-accelerating activities.
Main Methods:
- Homology modeling of CCPH was performed.
- Substitution mutagenesis was used to generate 24 CCPH mutants.
- Functional analyses were conducted on the generated mutants.
Main Results:
- Specific amino acids (R118, F144) are critical for cofactor activity (C3b/C4b inactivation).
- Other residues (R35, K142, K191) are essential for decay-accelerating activity.
- Mutations S100K and G110D significantly enhanced decay-accelerating activity without impacting cofactor function.
- Ionic interactions are key components of the CCPH binding interface.
Conclusions:
- The study successfully mapped critical functional sites within CCPH.
- CCPH utilizes specific amino acid residues for distinct complement regulatory functions.
- Enhanced decay-accelerating mutants offer potential for complement-targeted therapies.
Abstract:
Herpesvirus saimiri is known to encode a homolog of human complement regulators named complement control protein homolog (CCPH). We have previously reported that this virally encoded inhibitor effectively inactivates complement by supporting factor I-mediated inactivation of complement proteins C3b and C4b (termed cofactor activity), as well as by accelerating the irreversible decay of the classical/lectin and alternative pathway C3 convertases (termed decay-accelerating activity). To fine map its functional sites, in the present study, we have generated a homology model of CCPH and performed substitution mutagenesis of its conserved residues. Functional analyses of 24 substitution mutants of CCPH indicated that (i) amino acids R118 and F144 play a critical role in imparting C3b and C4b cofactor activities, (ii) amino acids R35, K142, and K191 are required for efficient decay of the C3 convertases, (iii) positively charged amino acids of the linker regions, which are dubbed to be critical for functioning in other complement regulators, are not crucial for its function, and (iv) S100K and G110D mutations substantially enhance its decay-accelerating activities without affecting the cofactor activities. Overall, our data point out that ionic interactions form a major component of the binding interface between CCPH and its interacting partners.
