Mechanisms of cell motility inhibition by the HIV-1 pathogenesis factor NEF
Final Report Abstract
Nef is a pathogenicity factor of HIV-1, HIV-2 and SIV that potently optimizes virus replication in the infected host and thus accelerates disease progression to AIDS in vivo. A large number of cellular interaction partners has been identified and the binding sites for some of these proteins have been mapped to distinct locations within Nef. By acting as a protein interaction adaptor, the viral protein manipulates a remarkable range of host cell processes, including signal transduction and vesicular transport pathways. However, the importance of the individual in vitro functions of Nef as well as the relevance of the proteins to the pathogenesis of the primate lentiviruses remain unclear. In addition, HIV-1 Nef was shown to interfere with the motility of infected CD4+ T cells. Our previous work identified that the underlying mechanism involves Nef’s ability to reduce host cell actin turnover via interaction with PAK2. Further, preliminary data suggested that Nef inhibits T cell polarity via an additional, actin-independent pathway. Therefore, our research during the past funding period focused on the characterization of Nef-mediated interference with T cell polarity and motility via an actin-independent mechanism. We defined the molecular determinants in Nef required for disruption of CD4+ T cell polarity and identified membrane association as well as the interaction surfaces for NAKC and PAK2 as important for this function of Nef. A second aim focused on the characterization of host cell components that are targeted by Nef to interfere with host cell actin dynamics and T cell motility. In this part of the study we confirmed binding of Nef to the newly identified cellular interaction partner EXOC2 and β-catenin. EXOC2 was shown to be critical for the disruption of actin remodeling and proximal TCR signaling, however did not play a role for Nefmediated effects on chemokine-induced actin dynamics or host cell vesicular transport. In contrast, we were not able to identify a significant role of ß-catenin in the Nef-mediated alteration of host cell actin dynamics and motility. In collaboration with Vérollet et al., we showed that Nef employs distinct and cell-type specific mechanisms to interfere with cell migration. Interestingly, Nef exerts opposing effects on the motility of the two main target cells of HIV: while Nef inhibits amoeboid migration of T cells, it enhances mesenchymal migration of macrophages via a mechanism that includes interaction with the myeloidspecific Src kinase Hck. Our previous results identified that Nef inhibits transendothelial migration (TEM) via an actinindependent pathways and another aim of the second funding period was to dissect the mechanisms that govern this Nef activity. Ex vivo analysis of Nef-expressing mouse T lymphocytes revealed that Nef reduced cell polarization and induced a mild reduction of diapedesis rates. Again, the NAKC interaction surface in Nef was identified as important for these processes. Finally, adoptive transfer experiments with mouse CD4+ T cells expressing Nef showed that homing to peripheral and mesenteric lymph nodes was markedly reduced compared to control cells. Our analysis of Nef mutant variants confirm our in vitro and ex vivo data that the interaction surface for PAK2 as well as NAKC play critical roles for the ability of Nef to interfere with CD4+ T lymphocyte migration in vivo. Together, these results suggest disruption of T cell polarization and extravasation as a novel mechanism by which Nef impairs T lymphocyte motility and homing to lymph nodes in vivo. Identification of two molecular surfaces in Nef that interfere with lymph node homing via two distinct mechanisms suggest that this activity may be critical for the role of Nef in AIDS pathogenesis and highlights Nef as potential target for antiviral therapy.
Publications
- (2012). HIV-1 Nef Interferes With T Lymphocyte Circulation Through Confined Environments in vivo. Proc. Natl. Acad. Sci. USA, 109: 18541–18546
Stolp, B., Imle, A., Coelho, F.M., Hons, M., Mendiz, R.G., Lyck, R., Stein, J.V. and Fackler, O.T.
(See online at https://doi.org/10.1073/pnas.1204322109) - (2014). Adding new dimensions: towards an integrative understanding of HIV-1 spread. Nature Reviews Microbiology, 12: 563–574
Fackler, O.T., Murooka T. T., Imle A., Mempel T.R.
(See online at https://doi.org/10.1038/nrmicro3309) - (2015). Association with PAK2 enables functional interactions of lentiviral Nef proteins with the exocyst complex. mBio 6(5): e01309-15
Imle, A., Abraham, L., Tsopoulidis, N., Hoflack, B., Saksela, K., Fackler, O.T.
(See online at https://doi.org/10.1128/mBio.01309-15) - (2015). HIV-1 reprograms the migration of macrophages. Blood, 125: 1611-1622
Vérollet, C., Souriant, S., Bonnaud, E., Jolicoeur, P., Raynaud-Messina, B., Kinnaer, C., Fourquaux, I., Imle, A., Benichou S., Fackler O.T., Poincloux, R., Maridonneau-Parini. I.
(See online at https://doi.org/10.1182/blood-2014-08-596775) - (2016). D186/D190 is an alleledependent determinant of HIV-1 Nef function. Virology, 498: 44-56
Imle, A., Stolp, B., Böhmer, V., Geyer, M., Raz, E., Fackler O.T.
(See online at https://doi.org/10.1016/j.virol.2016.08.012) - (2018). HIV-1 Nef Disrupts CD4+ T Lymphocyte Polarity, Extravasation, and Homing to Lymph Nodes via Its Nef-Associated Kinase Complex Interface. Journal of Immunology, 201:2731-2743
Lamas-Murua, M., Stolp, B., Kaw, S., Thoma, J., Tsopoulidis, N., Trautz, B., Ambiel, I.,Reif, T., Arora, S., Imle, A., Tibroni, N., Wu, J., Cui, G., Stein, J.V., Tanaka, M., Lyck, R., Fackler O.T.
(See online at https://doi.org/10.4049/jimmunol.1701420)
