Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation

Abstract

The N-terminal alpha-helix domain of the human immunodeficiency virus type 1 (HIV-1) Nef protein plays important roles in enhancement of viral infectivity, virion incorporation of Nef, and the down-regulation of major histocompatibility complex class I (MHC-I) expression on cell surfaces. In this study, we demonstrated that Met 20 in the alpha-helix domain was indispensable for the ability of Nef to modulate MHC-I expression but not for other events. We also showed that Met 20 was unnecessary for the down-regulation of CD4. These findings indicate that the region governing MHC-I down-regulation is proximate in the alpha-helix domain but is dissociated functionally from that determining enhancement of viral infectivity, virion incorporation of Nef, and CD4 down-regulation.

FIG. 1

Analysis of cellular expression of Nef proteins. (A) Sequences of HIV-1 nef mutants used here are shown. The N-terminal 45 residues of Nef and the corresponding nucleic acid sequence are depicted. The asterisk indicates the stop codon. (B and C) Lysates containing an equal amount of p24 capsid antigen were prepared from HeLa cells transfected with the respective proviral DNA clones and were analyzed by Western blotting with a rabbit anti-Nef antiserum (B) and a human anti-HIV-1 antiserum (C).

FIG. 2

Incorporation of Nef proteins into virions. HIV-1 WT and nef mutant viruses were prepared in HeLa cells transfected with the respective proviral DNA clones. Virus lysates containing an equal amount of p24 capsid antigen were analyzed by Western blotting with a rabbit anti-Nef antiserum (A) and a human anti-HIV-1 antiserum (B).

FIG. 3

Analysis of the infectivity of the WT and nef mutants of HIV-1. (A and B) Replication kinetics of the viruses in PBMCs. Viruses were obtained from HeLa cells transfected with WT and various nef mutant proviral clones. Unstimulated PBMCs (10⁵) were infected with 1 × 10⁵ (A) and 2 × 10⁴ (B) cpm of RT from viruses, and 2 days later, cells were stimulated by the addition of 0.5 μg of phytohemagglutinin P per ml. A half volume of the culture supernatants was harvested and refed with the same volume of culture medium with 50 U of recombinant human interleukin-2 per ml every 2 days. Kinetics of RT production in the culture supernatants are indicated. (C) Single-round infectivity of the viruses. Infectivity was determined by counting blue foci of X-Gal-treated MAGI cells 2 days after inoculation with the viruses. Averages and standard deviations of triplicate titrations of the same viral stock are shown.

FIG. 4

Effect of mutations in Nef protein on MHC-I down-regulation on the surfaces of HIV-1-infected cells. (A) CEM-GFP cells (10⁵) were infected with 5 × 10⁵ cpm of RT from each virus obtained from transfected HeLa cells, and 3 to 5 days later the cells were reacted with an RPE-labeled anti-MHC-I antibody at 4°C for 1 h. The cells were washed, fixed with 1% formaldehyde, and analyzed for fluorescence intensity by flow cytometry. (B) The level of MHC-I expression on the GFP-positive population in virus-inoculated CEM-GFP cells was evaluated as geometric mean fluorescence using CELLQuest software (Becton Dickinson).

FIG. 5

Effect of mutations in the Nef protein on CD4 down-regulation on the surfaces of cells infected with VSV-G–HIV-1 pseudotyped virus. The pseudotyped viruses were prepared by cotransfection of pCMV-G and pNL43-Ude1.K1 (B), pNL43-Ude1.K1.nM1T (C), pNL43-Ude1.K1.nM20A (D), or pNL43-Ude1.K1.nM20R (E) into HeLa cells. CEM-GFP cells (10⁵) were infected with 2.5 × 10⁷ cpm of RT from each pseudotyped virus, and 2 days later, cells were treated at 4°C for 1 h with RPE-labeled anti-MHC-I antibody and allophycocyanin-labeled anti-CD4 antibody. The cells were then washed, fixed with 1% formaldehyde, and analyzed for fluorescence intensity for CD4 and MHC-I in the cell population expressing GFP by flow cytometry.