Involvement of a C-terminal motif in the interference of primate lentiviral Vpu proteins with CD1d-mediated antigen presentation

Susanna M. Bächle1, Daniel Sauter2, Sabrina Sibitz1, Johan K. Sandberg1, Frank Kirchhoff2, Markus Moll1
Affiliates: 1Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden, 2Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany

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The HIV-1 accessory protein Vpu is emerging as a critical factor for viral evasion from innate immunity. We have previously shown that the Vpu proteins of two HIV-1 group M subtype B strains (NL4-3 and BaL) down-regulate CD1d from the surface of infected dendritic cells (DCs) to inhibit their crosstalk with the innate invariant natural killer T (iNKT) cells. The Vpu proteins of rare HIV-1 group N strains, however, interfere only poorly with CD1d. This suggests that the genetically diverse Vpu proteins from primate lentiviruses may differ in this immune evasion function and the structural determinants involved in CD1d interference. The aim of the present study was thus to determine the CD1d down-regulation capabilities of Vpu proteins from all four groups of HIV-1 and their vpuencoding SIV counterparts and to identify sequence motifs in Vpu required for CD1d interference.

HEK cells were co-transfected with human CD1d and the various vpu alleles and mutants. When possible findings were confirmed in a DC infection system. Transfected HEK cells were co-cultured with human iNKT cells to determine the effect of decreased CD1d surface levels on iNKT activation.

We found that the Vpu proteins of HIV-1 groups M, O, and P, as well as most of their simian precursors inhibited the expression of human CD1d, whereas those derived from rare HIV-1 group N strains were poor CD1d antagonists. Further analysis showed that this Vpu function was conserved among HIV-1 M subtypes with the notable exception of subtype C Vpu proteins. Importantly, the capacity to down-regulate CD1d correlated with the ability to inhibit iNKT cell activation. Mechanistically, the analysis of different mutants and chimeras derived from active subtype B and inactive subtype C Vpu proteins revealed that the cytoplasmic rather than the transmembrane domain of Vpu contains determinants involved in CD1d down-regulation. We identified a C-terminal APW motif specific for group M subtype B Vpu proteins necessary for interference with CD1d surface expression.

The results presented in this study show that the ability to interfere with CD1d surface expression and iNKT cell activation is broadly conserved between diverse primate lentiviral Vpu proteins and involves structural determinants located at the C-terminus of Vpu. Overall, the presented data further support a role for Vpu in lentiviral evasion from innate cell-mediated immunity.