Human immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro: the cytoplasmic domain of CD4 contributes to Vpu sensitivity

Abstract

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (H1V-i) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability ofVpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these

Chapter 2 Viral Channel-Forming Proteins

“…(1) Vpu is involved in the enhanced release of progeny virions (Klimkait et al, 1990) by directing the HIV receptor CD4 to the ubiquitindependent proteasome degradation pathway in the ER (Bour et al, 1995;Chen et al, 1993;Friborg et al, 1995;Margottin et al, 1996;Tiganos et al, 1997). The CD4-Vpu interaction is determined by the Vpu cytoplasmic domain Kimura et al, 1994;Margottin et al, 1996;Schubert et al, 1996a;Willey et al, 1992).…”

“…However, there is increasing evidence at least for Vpu that the protein interferes with several host membrane proteins at various locations within the cell. Since no feedback mechanism regulating Vpu production is known to date, it is reasonable to assume that Vpu can engage in these protein-protein interactions (CD4: Bour et al, 1995;Chen et al, 1993;Friborg et al, 1995;Kimura et al, 1994;Margottin et al, 1996;Schubert et al, 1996a;Tiganos et al, 1997;Willey et al, 1992;TASK: Hsu et al, 2004;BST-2: Neil et al, 2008;van Damme et al, 2008) but also can self-assemble forming channels or pores. The same situation counts for the other channel-forming proteins and needs to be elucidated in the future.…”

Cell type‐dependence for Vpu function

“…While Vpu is not required for HIV-1 replicaton in vitro [13,18,20], it has been shown to enhance the release of viral particles [9,13,20,24] and to disrupt complexes formed in the endoplasmic reticulum between gp160 and the cell surface receptor CD4 [14,21,22]. Vpu has alscj been shown to enhance the degradation of CD4 when coexpressed in HeLa cells [1,14,21-231. There are at least two possible general mechanisms for Vpu function.…”

“…In this study, we describe preliminary experiments undertaken to determine the effect of Vpu on virus production from Cos 1 cells. It was found that a mutation in Vpu previously shown to reduce viral titer by approximately 90% over one round of replication in HeLa cells [9] has no effect on virus production from Cos 1 cells. In addition, pulse/ chase analysis indicated that capsid protein (p24) release from Cos 1 cells was equivalent in the presence or absence of Vpu expression.…”

SGTA: A New Player in the Molecular Co-Chaperone Game

“…Human SGTA also interacts with HIV-1 VPU and the viral core protein precursor group-specific antigen (GAG) [14]. VPU facilitates rapid degradation of CD4, the HIV-1 receptor, in the endoplasmic reticulum [20,90] and enhances virus particle exit from the plasma membrane of infected A3.01 cells (a CD4 + lymphocytic cell line) [77,78]. GAG is important in virus assembly and maturation, is itself sufficient for the production of immature virus particles, and is involved in the recruitment of cellular proteins involved in viral assembly and budding [10,70].…”