Figure 2: Vpu compromises generation of MHC class I complexes in HIV-1 infected PBL. (A) Pulse/chase analysis of MHC-I molecules in TCD8+- depleted and PHA/IL-2–activated PBL infected with HIVNL4-3 (wild type, +Vpu), or the mutant HIVNL-U35 (−Vpu); uninfected control (mock). MHC class I complexes were immunoprecipitated with mAb W6/32, separated in a 10% acryl aide gel, and analyzed by fluorography. Only parts of the fluorograms demonstrating H chain–specific bands in the range of 43 kD are shown. (B) Relative amounts of H chain proteins seen in (A) were quantitated by means of an image analyzer and blotted against the time of the chase period (pu, nonstandardized arbitrary PhosphoImager units). (C) TCD8+depleted and PHA/IL-2–activated PBL isolated from another donor were infected with HIVNL4-3 (+Vpu), HIVNL-U35 (−Vpu), or HIVNL4-3/U2/6 expressing a nonphosphorylated mutant (+Vpu2/6). Pulse/chase experiment, immunocollection, and SDS-PAGE analysis (not shown) were performed as described for Fig. 2, A and B. Relative amounts of H chains were quantitated with a PhosphorImager and plotted in arbitrary units (pu) against the time of the chase period.

Mentions: To further characterize the effect of Vpu on the expression of MHC-I molecules, PHA/ IL-2–activated, TCD8+-depleted PBL from two donors were infected for 4 d with HIVNL4-3 or isogenic vpu mutants, and class I biogenesis was biochemically characterized (Fig. 2). After 45 min of radiolabeling with [35S]methionine and chasing for various times at 37°C, class I molecules reactive with a mAb specific for conformed class I molecules were collected, separated by SDS-PAGE, and quantitated. Cells from either donor infected with wild-type virus demonstrated a 30–40% reduction in the amount of class I molecules recovered at the initiation of the chase, compared to uninfected or vpu-deficient cultures (Fig. 2, B and C). Additionally, an augmented decay of class I H chains was detectable in the presence of wild-type Vpu after a 4-h chase period (Fig. 2 A, 2) relative to either uninfected cells (Fig. 2 A, 1), cells infected with the Vpu-deficient mutant HIVNL-U35 (Fig. 2 A, 3), or the Vpu phosphorylation mutant HIVNL4-3/U2/6 (Fig. 2 C). Note that the effects of Vpu on class I biosynthesis are underestimated in these experiments because not all cells are infected in each culture (Fig. 1 B, f–h). Consistent with the Vpu-dependent effects in PBL (Fig. 2), class I molecules were also affected in a Vpu-dependent manner in HIV-1–infected CD4+ T cell line A3.01 (not shown). Neither the amount of β2m recovered from cell lysates nor its secretion into the culture supernatant was altered by Vpu in HIV-1–infected PBL or T cells (not shown), indicating that Vpu does not prevent class I assembly by reducing β2m to limiting levels. Rather, we conclude that the Vpu-dependent downregulation of class I cell surface expression in cells infected with wild-type HIV-1 largely reflects Vpu-mediated instability of MHC-I molecules due to their improper assembly or enhanced degradation.

Kerkau T, Bacik I, Bennink JR, Yewdell JW, Húnig T, Schimpl A, Schubert U - J. Exp. Med. (1997)

Bottom Line: This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4.VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER.The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47.

Affiliation: Institute of Virology and Immunobiology, University of Würzburg, Germany.

Abstract: The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.