Long-Distance Tunneling Nanotubules Shuttle Viral Immunoglobulin Class Switch-Suppressing Factors from HIV-Infected Macrophages to B Cells.

Class switching from immunoglobulin M (IgM) to IgG, IgA or IgE is central to immunity against viruses and requires activation of B cells by CD4+ T cells through CD40 ligand (CD40L). Human immunodeficiency virus (HIV) evades protective T cell-dependent IgG and IgA responses against viral proteins and vaccines through mechanisms that remain unknown. Recent findings from our lab suggested the involvement of negative factor (Nef), an HIV accessory protein that suppresses CD40 signaling in B cells. The present work addressed the mechanism by which Nef invades B cells. Fluorescence microscopy-based studies showed that accumulation of Nef in infected lymphoid follicles was associated with severe loss of activation-induced cytidine deaminase (AID), a Nef-targeted DNA-editing enzyme essential for class switching. Loss of follicular AID correlated with in vivo inactivation of CD40 signaling and formation of long-distance membrane tethers connecting Nef-positive B cells with HIV-infected macrophages. By combining confocal microscopy, videomicroscopy and electron microscopy with standard molecular biology techniques, we demonstrated that macrophages harboring HIV or expressing a chimeric Nef-green-fluorescent protein were capable of transferring Nef-containing cargos to B cells via long-distance, actin-propelled and energy-dependent membrane channels, including tunneling nanotubules. Transfer of Nef to B cells involved a clathrin-dependent pathway that required the guanine nucleotide exchange factor Vav as well as downstream Rho, Rac and Ccd42 small GTPases. This pathway led to Nef-mediated inhibition of CD40-induced AID expression and IgG and IgA class switching. In summary, our findings indicate that HIV evades T cell-dependent IgG and IgA responses by shuttling virus-encoded Ig class switch-suppressing factors from infected macrophages to non-infected B cells via long-range membrane tethers, including tunneling nanotubules.