Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles

Abstract

Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA 98:12144-12149, 2001). These Nef functions could contribute to impaired CD4(+)-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether nef alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1 nef alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV) nef alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably, nef genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas nef alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.

FIG. 1.

Correlation between Nef and GFP expression. (A) HeLa-CIITA cells and Jurkat cells were transfected with a bicistronic vector coexpressing GFP and NL4-3 Nef (upper panel) or GFP alone (lower panel). As described in Materials and Methods, cells were permeabilized, and Nef and GFP expression was detected simultaneously by two-color flow cytometric analysis. Ranges for green fluorescence on cells defined as expressing no (N), low (L), medium (M), or high (H) levels of GFP are indicated. (B) Quantitation of Nef expression in the transiently transfected cells shown in panel A. The ordinate gives the mean channel numbers of red Nef fluorescence for the four different ranges of GFP expression.

FIG. 2.

Modulation of MHC-II and Ii cell surface expression by Nef is conserved between different groups of primate immunodeficiency viruses. (A) Flow cytometric analysis of HeLa-CIITA cells transfected with a bicistronic vector expressing GFP alone (NL4-3nef*) or GFP with either NA7, NL4-3, SIVmac239, or HIV-2 Ben Nef. Ranges for green fluorescence are indicated. (B) Quantitative effect of HIV-1 NA7, HIV-1 NL4-3, SIVmac239, HIV-2 Ben, and HIV-2 Rod Nef on MHC-II and Ii surface expression. Values were determined as described in Materials and Methods. Shown are data derived from one representative transfection. Similar results were obtained in three independent experiments.

FIG. 3.

Modulation of MHC-II and Ii cell surface expression is a conserved property of primary and consensus HIV-1 nef alleles. Quantitative presentation of MHC-II down-modulation (upper panel) and up-regulation of Ii (lower panel) by nef alleles obtained from progressors of HIV-1 infection (A), LTNPs (B), and consensus Nef sequences derived from a large number of primary nef alleles (C) (8, 38). Parameters were determined and reproduced in independent experiments, as described in the legend to Fig. 2.

FIG. 4.

Effect of Nef on MHC-II and Ii cell surface expression in HIV-1-infected cells. Human PBMC (upper panel), HeLa CIITA cells (middle panel), and 221-B7 cells (lower panel) were transduced with GFP-expressing HIV-1 NL4-3 particles containing an intact (nef⁺) or disrupted (nef*) nef gene pseudotyped with the VSV-G glycoprotein. FACS profiles were determined as described in Materials and Methods. The results were generated from the same pool of transduced cells. Similar results were obtained in independent experiments and when either the L243 or the TÜ36 antibody was used for MHC-II detection.

FIG. 5.

Functional analysis of HIV-1 NA7 Nef mutants. (A) The NA7 Nef amino acid sequence is given in single-letter code. Mutated positions are underlined and sites interacting with some previously defined Nef-binding proteins (20, 55) are indicated. HeLa-CIITA cells (B and C) or Jurkat T cells (D to F) were transfected with the indicated HIV-1 NA7 Nef mutants and assayed for surface expression of MHC-II (B), Ii (C), MHC-I (D), CD4 (E), and CD28 (F). Quantification was performed as described in Materials and Methods. The results were confirmed in two independent experiments.