Loss of Nef-mediated CD3 down-regulation in the HIV-1 lineage increases viral infectivity and spread

Abstract

Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef-and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-tocell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread.HIV | CD3 | Nef | T cell | Env

HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells

“…For RNAi knockdown of DCAF1, primary CD4+T cells were activated for 4 days with 1μg/ml plate-bound αCD3 antibody (cloneOKT3, Biolegend) in the presence of 2 μg/mL soluble αCD28 antibody(clone CD28.2, Biolegend). RNAi knockdown of DCAF1 was performed as described before 52 using ON-TARGET plusHuman DCAF1 siRNA - SMARTpool (Dharmacon) and non-targeting siRNA (Dharmacon) was used as a control. 2×10 6 cells were electroporated with 200 pmol siRNA using a NeonTransfection System (Thermo Fisher Scientific; three pulses, 10 ms, 1,600 V).…”

A Conserved Tryptophan in the Envelope Cytoplasmic Tail Regulates HIV-1 Assembly and Spread

“…Infected Jurkat cells were stained with envelope-specific antibodies PGT151 Ab (5 µg/mL) and 2G12 (5 µg/mL) for 60 min at 37 • C in FCS-supplemented RPMI to allow for Env internalisation [39]. Cells were thoroughly washed in ice-cold FACS wash buffer, and surface Env was detected with anti-human-PE secondary antibody for 30 min on ice to measure Env surface levels at T 0min.…”

“…WTand W757A-infected T cells (Jurkat and primary CD4 + T cells) expressed identical levels of Env on the surface of infected cells when stained with either the Env-specific mAbs PGT151 (for the gp120:gp41 interface and functional timers) (Figure 5C) or 2G12 (recognising a non-conformation-dependent carbohydrate epitope on gp120) (Figure 5D). Once at the PM, a highly conserved membrane-proximal AP-2 binding motif (YxxL) acts to limit the amount of Env presented at the surface of infected cells with internalised Env being recycled back to the PM [13][14][15][16][17]20,39]. To test the recycling kinetics of WT and W757A Env, we used a dual-fluorophore flow cytometry-based assay [39,50].…”

The envelope cytoplasmic tail regulates HIV-1 assembly and spread

“…Once at the PM, a highly conserved membrane proximal AP-2 binding motif (YxxL) acts to limit the amount of Env presented at the surface of infected cells with internalised Env being recycled back to the PM (Mesner et al, 2020;Groppelli et al, 2014;Kirschman et al, 2017). To test the recycling kinetics of WT and W757A Env we used a dual-fluorophore flow cytometry-based assay (Anand et al, 2019;Mesner et al, 2020). Two Env antibodies, 2G12 and PGT151 were allowed to bind surface Env at 4 0 C and detected using a PE-conjugated secondary antibody.…”

“…Infected Jurkat cells were stained with Envelope specific antibodies PGT151 Ab (5 μg/mL), and 2G12 (5 μg/mL) for 60 min at 37°C in FCS supplemented RPMI to allow for Env internalisation (Mesner et al, 2020). Cells were thoroughly washed in ice cold FACS wash buffer and surface Env was detected with anti-human-PE secondary antibody for 30 min on ice to measure Env surface levels at T0min.…”