Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS

Abstract

The human immunodeficiency virus type 1 (HIV-1) Nef protein has several independent functions that might contribute to efficient viral replication in vivo. Since HIV-1 adapts rapidly to its host environment, we investigated if different Nef properties are associated with disease progression. Functional analysis revealed that nef alleles obtained during late stages of infection did not efficiently downmodulate class I major histocompatibility complex but were highly active in the stimulation of viral replication. In comparison, functional activity in downregulation of CD4 and enhancement of HIV-1 infectivity were maintained or enhanced after AIDS progression. Our results demonstrate that various Nef activities are modulated during the course of HIV-1 infection to maintain high viral loads at different stages of disease progression. These findings suggest that all in vitro Nef functions investigated contribute to AIDS pathogenesis and indicate that nef variants with increased pathogenicity emerge in a significant number of HIV-1-infected individuals.

FIG. 1

Activity of nonprogressor and progressor consensus nef alleles. (a) Amino acid variations between NP and P consensus Nef sequences. Numbers refer to the corresponding position in the NL4-3 Nef amino acid sequence. (b) Flow cytometric analysis (upper panel) and quantitative effect of the indicated nef alleles on CD4 and class I MHC cell surface expression (lower panel). Flow cytometric analysis was performed, and CD4 and class I MHC expression was determined, as described in Materials and Methods. Ranges for green fluorescence on cells defined as expressing no (N), low (L), medium (M), or high (H) levels of GFP are indicated. (c) MAGI cells were infected in triplicate with aliquots of three different 293T cell-derived virus stocks containing 125 ng of p24 core antigen. Infectivity is shown relative to HIV-1 NL4-3 wild type. Error bars correspond to standard deviation. (d) Replication of the NL4-3 variants containing the indicated nef alleles (for symbols, see panel a) in PBMC. Cells were infected immediately after infection and stimulated with phytohemagglutinin 3 days later. All results were confirmed in at least three independent experiments. PSL, photostimulated light emission.

FIG. 2

Inefficient Nef-mediated class I MHC downregulation after disease progression. (a) Flow cytometric analysis and quantitative presentation of CD4 and class I MHC downregulation by nef alleles obtained from patients P2 and SP7 prior (87 and 88 samples) and during or after disease progression (91 and 93 samples). (b and c) Viral infectivity (b) and enhancement of viral replication by P2 and SP7 nef alleles (c). Parameters were determined and reproduced in independent experiments, as described in the legend to Fig. 1.

FIG. 3

nef alleles derived from a long-term nonprogressor are selectively impaired in CD4 downmodulation and stimulation of viral replication. (a) Structure of the chimeric and mutant Nef proteins that were analyzed (30). (b) Effect of the LTNP4-91-B1 nef allele on CD4 and class I MHC cell surface expression. (c to f) Functional activity of the indicated mutant and chimeric nef alleles in downregulation of CD4 (c), class I MHC downmodulation (d), enhancement of viral infectivity (e), and stimulation of HIV-1 replication in PBMC (f). Symbols are shown in panel a. For all four in vitro assay systems for Nef function, similar results were obtained in two to four independent experiments. Error bars shown in panel e represent standard deviation.

FIG. 4

Functional differences between nef alleles representing different stages of HIV-1 infection. Functional activity of a consensus nonprogressor nef allele (NPex), primary alleles obtained during the asymptomatic stage of infection (P2-87 and SP7-88), and the average activities obtained from the first samples drawn from the six patients in Fig. 4 (Pat E) were assigned a value of unity. The functional activity of other nef alleles is shown relative to these forms. Values for CD4 (at medium GFP expression levels) and class I MHC (at high GFP expression levels) downregulation were obtained from three to six independent experiments. Values obtained for the enhancement of viral replication were derived from four to six infections and represent relative reverse transcriptase activities measured at the peak activity of the NL4-3 wild-type strain, which was observed between 9 and 13 days postinfection. Infectivity values represent at least nine measurements with three independent virus stocks. Error bars give the standard deviations.

FIG. 5

Functional activity of primary nef alleles obtained at different stages of infection. The x-axis gives the number of CD4⁺ cells per microliter at the time of PBMC sampling. A total of 20 samples derived from eight patients (SP7, SP8, P2, P5, P7, P8, P9, and P10 (Tables 1 and 2) were analyzed. Average values (■) for downregulation of CD4 (at medium GFP expression levels) and class I MHC (at high GFP expression levels), as well as enhancement of HIV-1 replication and infectivity, are indicated. Values were determined as described in Materials and Methods and in Table 2.

FIG. 6

Schematic model for the modulation of independent Nef functions during the course of HIV-1 infection. The black bars indicate the changes in Nef function observed during progression to AIDS. In immunocompetent hosts, Nef efficiently downmodulates class I MHC to prevent CTL lysis of infected cells. After the breakdown of the immune system, the selective forces drive variations in Nef that are associated with an increased activity in the stimulation of HIV-1 replication and reduced functional activity in class I MHC downregulation. The ability of Nef to downregulate CD4 and to enhance virion infectivity is enhanced in some AIDS patients and is likely to optimize viral spread in a host environment where uninfected CD4⁺ target cells become limited. The acute phase of infection has not been investigated in this study. Symbols: ↓, downmodulation; ↑, enhancement.