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

Abstract

Nef is a multifunctional viral protein that has the ability to downregulate cell surface molecules, including CD4 and major histocompatibility complex class I (MHC-I) and, as recently shown, also members of the serine incorporator family (SERINC). Here, we analyzed the impact of naturally occurring mutations in HIV-1 Nef on its ability to counteract SERINC restriction and the clinical course of infection. HIV-1 Nef sequences were obtained from 123 participants of the Amsterdam Cohort Studies and showed multiple amino acid variations and mutations. Most of the primary Nef proteins showed increased activity to counteract SERINC3 and SERINC5 as compared to NL4-3 Nef. Several mutations in Nef were associated with either an increased or decreased infectivity of Bal26-pseudotyped HIV-1 produced in the presence of SERINC3 or SERINC5. The 8R, 157N and R178G Nef mutations were shown to have an effect on disease progression. Survival analysis showed an accelerated disease progression of individuals infected with HIV-1 carrying arginine or asparagine at position 8 or 157 in Nef, respectively, or the R178G Nef mutation. Here, we observed that naturally occurring mutations in Nef affect the ability of Nef to counteract SERINC3- and SERINC5-mediated inhibition of viral infectivity. The majority of these Nef mutations had no significant effect on HIV-1 pathogenesis and only the 8R, 157N and R178G mutations were associated with disease course.

Keywords: HIV-1; Nef; SERINC3; SERINC5; amino acid variations; disease progression; naturally occurring mutations.

Figure 1

Amino acid variation in Nef. HIV-1 consensus B Nef. The red boxes indicate previously identified amino acid polymorphisms [14,25,26]. The green boxes highlight known binding sites of Nef in which naturally occurring variations and amino acid polymorphisms occur.

Figure 2

(A) Downregulation of serine incorporator 3 (SERINC3) and SERINC5 membrane expression by HIV-1 Nef. HEK293T cells were co-transfected with plasmids expressing Nef and SERINC3 or SERINC5 or in combination with a relevant empty vector. The surface expression of SERINC3 and SERINC5 (orange) or in combination with HIV-1 Nef (red) was determined by flow cytometry as the geometric mean of fluorescent intensity. Background fluorescent (blue) control was included. (B) Downregulation of SERINC3 and SERINC5 by Nef as shown as relative expression as compared to the control of four independent experiments (p < 0.05). (C) The effect of SERINC3 (left panel) and SERINC5 (right panel) on HIV-1 infectivity: Infectivity of Bal26-pseudotyped HIV-1 produced in 293T cells overexpressing Nef (black bar), SERINC3 or SERINC5 (dark gray bar) and SERINC3 or SERINC5 in combination with Nef (light gray bar) in U87 CD4+CCR5+. Infectivity was determined by luciferase activity in relative light units (RLU). Mean ± SD are shown. (D) Expression of Nef from pcDNA3.1 encoding primary Nef proteins and β-actin levels were determined by Western blotting.

Figure 3

Amino acid variation in Nef proteins obtained from individuals with HIV-1. HIV-1 consensus B Nef, NL4-3 Nef (Nef1) and Nef proteins from patients with HIV-1 (Nef5-24). Red boxes indicate some of the previously described amino acid polymorphisms [14,25,26] and binding sites for reference.

Figure 3

Amino acid variation in Nef proteins obtained from individuals with HIV-1. HIV-1 consensus B Nef, NL4-3 Nef (Nef1) and Nef proteins from patients with HIV-1 (Nef5-24). Red boxes indicate some of the previously described amino acid polymorphisms [14,25,26] and binding sites for reference.

Figure 4

Effect of naturally occurring amino acid variations on infectivity of Bal26-pseudotyped HIV-1. Infectivity of Bal26 Nef-deficient single-round luciferase reporter virus produced in 293T cells overexpressing SERINC3 (A) or SERINC5 (B) in the presence of wild-type NL4-3 Nef (Nef1) or Nef proteins obtained from individuals with HIV-1 (Nef5-Nef24). The infectivity of the virus produced in the presence of the Nef and SERINC3/5 was corrected for the infectivity of the virus produced in the presence of the same primary Nef and an empty vector. Virus infectivity is expressed relative to the virus produced in the presence of NL4-3 Nef (fold change). Means ± SD of three independent experiments are shown. Unpaired two-tailed t-test (* p < 0.05; ** p < 0.01). (C) Downregulation of SERINC5 membrane expression by primary Nef proteins was determined by flow cytometry (Left panel—orange: Nef1; red: Nef24; blue: background fluorescent control). Downregulation of SERINC5 membrane expression by primary Nef (Nef6, 14, 15, 18, and 24) relative to NL4-3 Nef (Nef1) (right panel). (D) Correlation between the ability of the primary Nef proteins to counteract SERINC3 and SERINC5.

Figure 5

Effect of naturally occurring mutations in Nef on HIV-1 infectivity. The ability of primary Nef proteins that do or do not contain the indicated amino acids or mutations to counteract (A) SERINC3- or (B) SERINC5-mediated HIV-1 restriction was compared. Virus infectivity is expressed relative to the virus produced in the presence of NL4-3 Nef (fold change of three independent experiments). Means ± SD of Nef proteins containing the indicated amino acid or mutation are given. Mann–Whitney U test (* p < 0.05; ** p < 0.01). Black bar: indicated amino acid or mutation is absent; grey bar: indicated amino acid or mutation is present.

Figure 6

Effect of naturally occurring mutations in Nef on disease progression. Kaplan–Meier survival analysis for amino acid variations or mutations 8R (A), 157N (B) and R178G (C) in Nef with time in months from seroconversion to progression to AIDS as defined by the CDC definition 1993 (top panel) or AIDS-related death (bottom panel). P values displayed represent log-rank test.