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. 2001 Jul;75(13):6173-82.
doi: 10.1128/JVI.75.13.6173-6182.2001.

Differential incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation

M T Esser  1 , D R Graham, L V Coren, C M Trubey, J W Bess Jr, L O Arthur, D E Ott, J D Lifson
Affiliations

Differential incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation

M T Esser et al. J Virol. 2001 Jul.

Abstract

Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4(+) T cells long before a quantitative decline in circulating CD4(+) T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4(+) T cells remains unclear. Both direct effects of cytopathic infection of CD4(+) cells and indirect effects in which uninfected "bystander" cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.

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Figures

FIG. 1
FIG. 1
Survey analysis of differential incorporation of CD80, CD86, MHC-I, and MHC-II into virions of a panel of HIV-1 isolates propagated in PBMC, MΦ, or cell lines. Immunoprecipitation of primary HIV-1 isolates, MΦ-tropic isolates, and cell line-adapted virions was performed by using a MAb-based VPA as described in Materials and Methods. MAbs to immunoregulatory proteins CD80, CD86, MHC-I, and MHC-II were used to characterize primary isolates (108-436, 2-285, P419, 115, 92US657, 92US727, and 91US054 [blue shades]), MΦ isolates (Ada-M 98-4 and Ba-L 98-6 [red shades]) and cell line-adapted isolates (MN/H9, NL4-3/T2, and NL4-3/TBLCL-CD4 [green shades]). A polyclonal antiserum raised against microvesicles derived from the TBLCL-CD4 cell line served as a positive control for maximal virion precipitation, and an isotype-matched mouse anti-CD4 MAb served as a negative control. The data shown are representative of two independent experiments performed in triplicate with <10% variability in the magnitude of virion clearance. Error bars represent 1 standard deviation of the mean of triplicate measurements. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student's t-test significance of differences between experimental measurements and isotype control measurements).
FIG. 2
FIG. 2
Profile of immunoregulatory molecules incorporated into HIV-1 virions produced from infected MΦ. MΦ were isolated and cultured as described in Materials and Methods. MΦ were mock infected or infected with Ada-M 98-3, Ba-L 98-4, or Ba-L 98-7. MΦ- derived virions were characterized for the presence of CD4, CD45, CD55, CD80, CD86, MHC-I, and MHC-II in the virion envelope. The data shown are representative of two separate experiments, each performed in triplicate. Error bars represent 1 standard deviation of the mean of triplicate measurements. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student t-test significance of differences between experimental measurements and isotype control measurements). Microves., microvesicles.
FIG. 3
FIG. 3
Profile of immunoregulatory molecules incorporated into HIV-1 virions produced from infected PBMC. PBMC were isolated and cultured as described in Materials and Methods. PHA- and IL-2-activated PBMC were mock infected or infected with CCR5-tropic SF162, dual-tropic 89.6, or CXCR4-tropic NL4-3. PBMC-derived virions were characterized for the presence of CD4, CD45, CD55, CD80, CD86, MHC-I, and MHC-II in the virion envelope. The data shown are representative of two separate experiments, each performed in triplicate. Error bars represent 1 standard deviation of the mean of triplicate measurements. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student t-test significance of differences between experimental measurements and isotype control measurements). Microves., microvesicles.
FIG. 4
FIG. 4
Virion and microvesicle preparations contain high levels of CD45, CD55, CD86, MHC-I, and MHC-II but not CD80. Virions and microvesicles derived from the T1, T2, TBLCL-CD4, and H9 cell lines were purified by sucrose density gradient ultracentrifugation. TBLCL-CD4 cell lysates served as a positive control and a way to determine the sensitivities of the different antibodies in the Western blot assays. Virion and microvesicle preparations (50 μg of total protein per lane) and the TBLCL-CD4 lysates were analyzed on an SDS–5 to 20% nondenaturing polyacrylamide gel under reducing or nonreducing conditions. Immunoblots were probed with a MAb to CD45 (HI30), a polyclonal serum to CD55 (H-319), a goat polyclonal serum to CD80 (N-20), a MAb to CD86 (IT2.2), a MAb to MHC-I, and a MAb to MHC-II (L243). The results shown are representative of at least three independent Western blot assays for each protein.
FIG. 5
FIG. 5
Profile of immunoregulatory molecules incorporated into HIV-1 virions produced from chronically infected continuous cell lines. Chronically infected cell lines were maintained in culture as described in Materials and Methods. Cell-free HIV-1NL4-3 derived from the T1, T2, TBLCL-CD4, and H9 cell lines was purified by sucrose density gradient ultracentrifugation. The purified virion preparations were characterized for the presence of CD4, CD45, CD55, CD80, CD86, MHC-I, and MHC-II in the virion envelope. The data shown are representative of three separate experiments, each performed in triplicate. Error bars represent 1 standard deviation of the mean of triplicate measurements. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student t-test significance of differences between experimental measurements and isotype control measurements). Microves., microvesicles.
FIG. 6
FIG. 6
Virion-associated cellular molecules play a role in virion-triggered cell death. The effect of virion-associated, host cell-derived molecules in HIV pathogenesis was examined by using conformationally authentic, noninfectious HIV-1 virions. Noninfectious, Aldrithiol-2-inactivated virions (p24CA equivalents at 50 ng/ml) or microvesicles (total protein at 10 μg/ml) were used to pulse resting PBMC from a healthy, HIV-seronegative donor. After 10 days, the PBMC were enumerated for total cell numbers and percent viability by trypan blue analysis. The data shown are representative of three separate experiments. Error bars represent 1 standard deviation of the mean of triplicate measurements. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student t-test significance of differences between experimental measurements and PBS [control] measurements).
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