Session topic


Title: Mutational analysis of a putative heparan sulfate-binding domain in the HERV-K(HML-2) envelope
ID: PS 23
Type: Poster session
Talk time: 3 + 2 min
Session: Poster session 1
Receptors and entry

Speaker: Alaa Ramadan (Berlin/DE)

Abstract - Text

Abstract text (incl. references and figure legends)

Introduction: The endogenous retrovirus HERV-K(HML-2) is the most recently endogenized virus family in the human genome. Its RNAs and functional proteins are expressed and assemble into particles. In a previous study it has been shown that entry of this virus is mediated by interactions between its envelope protein and cell surface molecules including heparan sulfates (Hs).

Objective: This study aims to investigate the role of a putative Hs-binding domain in the envelop protein that is at a corresponding position to the heparin binding site in the envelope protein of the related Mouse Mammary Tumor Virus (MMTV).

Methods: A reconstituted HERV-K113 envelope version that allows regular protein expression, particle incorporation and entry into target cells was used. Site-directed mutagenesis was performed to convert positively charged amino acids (arginine or lysine) to alanine. Cells were transfected with each env encoding plasmid for expression and for production of pseudotyped HIV viral reporter particles. Entry assay was assessed by infecting cells with HERV-env (mutant) luciferase viruses. Immunoprecipitation by heparin beads was performed and Envs were detected by WB.

Result: All of the mutant envelopes showed similar expression to that of the wild type envelope protein (the reconstituted HERV-K113 envelope) and were incorporated into virus particles with similar efficiency. Immunoprecipitation of viruses by heparin beads showed that the K223A/K225A mutant envelope failed to bind heparin beads almost completely, but the single site mutant K225A envelope virus retained the ability to bind heparin. All of the other analyzed mutations in the corresponding MMTV Hs-binding site showed similar or even stronger binding comparing to the wild type envelope. However, all mutants, including the K223A/K225A, showed comparable level of virus entry.

Conclusion: Although the K223A/K225A mutant envelope showed a strongly reduced affinity for heparin beads, this mutant envelope nevertheless mediates efficient entry. This result indicates that other Hs at the cell surface might efficiently compensate for the lack of heparin mediated attachment by interaction with alternative sites of the envelope. Further investigation is recommended.