Impacts of epitope expression kinetics and class I downregulation on the antiviral activity of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Abstract

The determinants of CD8(+) cytotoxic T-lymphocyte (CTL) antiviral activity against human immunodeficiency virus type 1 (HIV-1) remain poorly defined. Although recent technological advances have markedly enhanced the ability to detect HIV-1-specific T cells, commonly used assays do not reveal their direct interaction with virus. We investigated two determinants of CTL antiviral efficiency by manipulating HIV-1 and measuring the effects on CTL suppression of viral replication in acutely infected cells. Translocation of a Gag epitope into the early protein Nef markedly increased the activity of CTL recognizing that epitope, in comparison to HIV-1 expressing the epitope normally in the late protein Gag. Because this epitope translocation resulted not only in earlier expression but also in loss of major histocompatibility complex class I downregulation by Nef, the activities of CTL against a panel of viral constructs differing in kinetics of epitope expression and class I downmodulation were compared. The results indicated that both the timing of epitope expression and the reduction of class I have profound effects on the ability of CTL to suppress HIV-1 replication in acutely infected cells. The epitope targeting of CTL and viral control of class I therefore likely play important roles in the ability of CTL to exert pressure on HIV-1.

FIG. 1.

Kinetics of Nef, Gag, and RT expression in acutely infected cells. T1 cells were acutely infected with NL4-3 and real-time quantitative reverse transcription-PCR was performed to measure the production of Gag-Pol and Nef transcripts over time.

FIG. 2.

Construction of HIV-1 with a transposed SL9 epitope in Nef and knockout of SL9 in Gag. The half genomes of modified HIV-1 NL4-3 contained in plasmids p83-2.1 and p83-10 are shown. Wild-type versions of each plasmid contain the SL9 epitope in Gag and unmodified Nef (last 10 amino acids shown), respectively. The SL9x mutation was introduced by altering the codon for the HLA A2 anchor residue from leucine to isoleucine in p83-2.1. The NefSL9 mutation was created by inserting sequences coding for 10 amino acids including the SL9 epitope into the 3′ end of nef.

FIG. 3.

Growth curves for HIV-1 containing Gag and Nef mutations. Virions produced by cotransfecting these plasmid constructs were evaluated for their growth after acute infection of T1 cells. The growth curves reflect the mean of two independent experiments (error bars represent one standard deviation in log₁₀ units). Viral growth, as reflected by the slope of virion concentration, was similar for the three viruses.

FIG. 4.

Enhancement of antiviral activity of SL9-specific CTL by expression of epitope in the nef reading frame. HIV-1 containing combinations of unmodified or translocated SL9 was tested for its inhibition by SL9-specific CTL. Target cells acutely infected with the indicated viruses were cocultured with an SL9-specific CTL clone at an effector-to-target cell ratio of 1:8 (a condition in which suppression of the wild-type virus was inefficient). (A to C) Viral replication in the presence or absence of CTL is shown as p24 antigen production over time. (D) CTL-mediated suppression of replication in log₁₀ units at each time point is plotted. All data are given as the mean of triplicates, and error bars represent one standard deviation. This experiment is representative of three independent experiments.

FIG. 5.

MHC-I downregulation in cells infected by virus containing the NefSL9 constuct. T1 cells were acutely infected with virus from which Nef was deleted (A), wild-type virus (B), or virus containing the NefSL9 mutation (C), each containing a reporter gene in the vpr reading frame. Flow cytometry was performed and infected cells were identified by gating on the reporter; histograms demonstrating the expression of the HLA on infected cells are shown. Background isotype antibody staining is shown in the open histograms, and specific HLA A2 staining is shown in the shaded histograms; the net fluorescence intensity (above isotype) is indicated. These data are representative of two independent experiments.

FIG. 6.

Antiviral activity of SL9-specific CTL against virus containing the Nef-SL9 construct or Nef deletion. Suppression of replication of a panel of HIV-1 mutants was assessed as in Fig. 4 above. Two time points are plotted. The status of SL9 expression and MHC-I downregulatory function is indicated below the bar for each virus. These data are representative of two independent experiments.