Multifunctional Roles of the N-Terminal Region of HIV-1 _SF2 Nef Are Mediated by Three Independent Protein Interaction Sites

Abstract

HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1SF2Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread. IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1SF2Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.

Variation in HIV-1 Nef function within and among viral subtypes reveals genetically separable antagonism of SERINC3 and SERINC5

“…Our finding that Nef-mediated antagonism of SERINC3 displayed a distinct functional hierarchy among viral subtypes compared to that of SERINC5 indicates that these two closely related Nef activities are in fact functionally separable and further suggests that in vivo selection pressures to maintain these two Nef activities might differ. Indeed, the majority of subtype B Nef clones internalized SERINC5 efficiently but were impaired in their ability to counteract SERINC3, whereas SERINC3 antagonism activity was maintained in most Nef clones derived from subtypes A, C and D. We also discovered that these differences in SERINC3 antagonism could largely be attributed to residues in Nef's N-terminal anchor region, which allows the protein to interact with the inner leaflet of the plasma membrane as well as a variety of cellular kinases that are critical to its function [ 45 ]. In particular, we demonstrated that polymorphisms at residues 8 and 11 reduced Nef’s ability to co-localize with SERINC3 (but not SERINC5) based on PLA staining.…”

HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

“…To gain insight into the molecular mechanism by which NEF impairs CD4 T‐cell help, we analyzed the activity of a series of mutant NEF proteins in the adoptive transfer model (Fig 3A). This included NEF F195A which lacks the ability to associate with the cellular p21‐activated kinase 2 (PAK2) to negatively modulate host cell actin dynamics and motility (O'Neill et al , 2006; Stolp et al , 2009; Stolp et al , 2012), a NEF variant with disrupted di‐leucine motif (NEF LLAA) that lacks the ability to internalize cell surface receptors such as CD4 (Craig et al , 1998; Greenberg et al , 1998), NEF AxxA in which an SH3 domain‐binding PxxP motif is disrupted and fails, e.g., to relocalize the TCR proximal kinase Lck from the plasma membrane to intracellular compartments (Saksela et al , 1995; Pan et al , 2012), as well as NEF variants carrying a deletion of an N‐terminal protein interaction platform (NEF ∆12‐39) or a mutation in an interaction motif within residues 12‐39 (NEF A32‐39) required for CD4 downregulation and SERINC5 antagonism (Ananth et al , 2019). Among these NEF mutants, only deletion/mutation of the motifs in the NEF N‐terminus impaired the ability of the viral protein to disrupt HEL‐specific antibody production (Fig 3B, Appendix Fig S1A).…”

“…The coding sequences for NEF SF2 A32‐39, NEF SF2 LLAA, or patient‐derived nef genes C122 and CB76 were cloned into pSTITCH using the restriction sites AgeI and PacI in a multiple‐cloning site that was previously subcloned along with IRES∆NGFR from pQCXIX backbone. The proviral constructs used are based on HIV‐1 NL4‐3 and either lack NEF expression (HIV‐1 ∆NEF), or encode for wild‐type NEF from HIV‐1 SF2 (HIV‐1 WT) or specific NEF mutants (Fackler et al , 2006; Ananth et al , 2019).…”

“…A predominant sequence that resembles the bulk population was then cloned into pEGFP‐N1 vector as Nef.GFP fusion protein (Galaski et al , 2016) or pSTITCH.IRES.∆NGFR vector (Stolp et al , 2012) for further in vitro or in vivo characterization, respectively. These patient‐derived Nefs were assessed for downregulation of cell surface CD4 and MHC‐I, inhibition of chemotaxis toward stromal cell‐derived factor‐1 alpha (SDF‐1α), and inhibition of anti‐CD3 induced peripheral F‐actin ring formation as described in detail previously (Stolp et al , 2009; Imle et al , 2015; Ananth et al , 2019). The isolated nef sequences are deposited in GenBank (NCBI, NIH) under the following accession numbers: MT223941 and MT223942.…”

“…Virus supernatant was harvested 48 and 72 h after transfection and filtered using 0.45 µm pore size. Virus was concentrated using 20% sucrose cushion (r min 43,000 g r max 98,000 g; SW 32 Ti Rotor at 24,000 rpm; 1.5 h), titers were determined by SYBR Green I-based PERT (SG-PERT) or p24 ELISA, and infectivity was evaluated by infection of Tzm-bl reporter cells (Ananth et al, 2019).…”