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

Abstract

HIV Nef counteracts cellular host restriction factors SERINC3 and SERINC5, but our understanding of how naturally occurring global Nef sequence diversity impacts these activities is limited. Here, we quantify SERINC3 and SERINC5 internalization function for 339 Nef clones, representing the major pandemic HIV-1 group M subtypes A, B, C and D. We describe distinct subtype-associated hierarchies for Nef-mediated internalization of SER-INC5, for which subtype B clones display the highest activities on average, and of SERINC3, for which subtype B clones display the lowest activities on average. We further identify Nef polymorphisms that modulate its ability to counteract SERINC proteins, including substitutions in the N-terminal domain that selectively impair SERINC3 internalization. Our findings demonstrate that the SERINC antagonism activities of HIV Nef differ markedly among major viral subtypes and between individual isolates within a subtype, suggesting that variation in these functions may contribute to global differences in viral pathogenesis.

Nef Obtained from Individuals with HIV-1 Vary in Their Ability to Antagonize SERINC3- and SERINC5-Mediated HIV-1 Restriction

“…Recent reports demonstrated that three specific sites in Nef, the amino acids 12 to 39, the ExxxLL motif (S163C) and the AP-2 binding site (R178G), were identified to be involved in Nef function [ 14 , 15 , 16 , 24 ] ( Figure 1 ). In addition, multiple amino acid variations in Nef (subtype B HIV-1) were identified to play a role in SERINC3 and SERINC5 antagonism [ 14 , 25 , 26 ] ( Figure 1 ) including 8R, 9S, 11P, 12G, 14A/P/S, 15A, 21K/R, 28E, 43I, N51T, 54D, 63E, 81F, H116N, 120F, V148L/X, 157N, 158K, 161N, M168I/X, 182E and 188S, of which some specifically affected SERINC3 (8R, 9S, 11P, 12G, 14A, 15A, 21K, 28E, 43I, 182E and 188S) or SERINC5 (14S, 21R, 54D, 63E, 81F, 120F, 157N and 158K) [ 26 ]. We analyzed primary Nef proteins obtained from 123 individuals with HIV-1 participating in the Amsterdam Cohort Studies for amino acid changes at the previously described positions and regions as compared to consensus B ( Supplementary Table S1 ).…”

“…We selected 20 primary Nef proteins from individuals with HIV-1 participating in the Amsterdam Cohort Studies that show amino acid variation in the AP-2 binding site, the ExxxLL motif, changes in the number of positively charged amino acids and length of the protein interaction site consisting of the amino acid residues 12–39 (AA 12–39) and contain the previously identified amino acid polymorphisms [ 14 , 25 , 26 ] ( Figure 3 ). The primary Nef proteins were cloned into expression plasmids and Nef expression by these constructs was demonstrated by Western blotting ( Figure 2 D).…”

“…Next, we determined whether changes in the length and charge of the protein interaction site and naturally occurring Nef mutations were associated with changes in Nef activity to counteract SERINC3 and SERINC5 ( Figure 5 ; all data are shown in Supplementary Table S2 ). Previous reports indicated that some naturally occurring mutations in Nef could affect both SERINC3 and SERINC5 (N51T, H116N, V148L/X, 161N and M168I/X), while others only affected SERINC3 (8R, 9S, 11P, 12G, 14A, 15A, 21K, 28E, 43I, 182E and 188S) or SERINC5 (14S, 21R, 54D, 63E, 81F, 120F, 157N and 158K) [ 14 , 25 , 26 ]. The change of charge in the protein interaction site (AA 12–39) and the presence of amino acids 12G, N51T, H116N and 188S in Nef were associated with a significant increased infectivity of Bal26-pseudotyped HIV-1 produced in the presence of SERINC3, while increased length of the protein interaction site (AA12–39), 9S, 43I and S163C also increased and 11P, 14A, V148X, 161N and R178G decreased infectivity, albeit not significantly ( Figure 5 A and Supplementary Table S2 ).…”

“…However, no effect of the charge changes was observed on HIV-1 disease progression. Several specific amino acids located in and near the protein interaction site in Nef have been described to affect the ability of Nef to downregulate SERINC3/5 [ 26 ]. We observed that glycine at position 12 and serine at position 188 of Nef were associated with increased infectivity, indicating an increased ability to downregulate SERINC3 and confirming previous observations [ 26 ].…”

“…Several specific amino acids located in and near the protein interaction site in Nef have been described to affect the ability of Nef to downregulate SERINC3/5 [ 26 ]. We observed that glycine at position 12 and serine at position 188 of Nef were associated with increased infectivity, indicating an increased ability to downregulate SERINC3 and confirming previous observations [ 26 ]. However, the effect of the other mutations in this region or in close proximity (AA8-43) on either SERINC3 and/or SERINC5 downregulation could not be confirmed.…”