The human immunodeficiency virus type 1 Vpu protein: a potential regulator of proteolysis and protein transport in the mammalian secretory pathway.
HIV-1 Vpu is a small transmembrane phosphoprotein of 16 kDa which performs critical roles in CD4 proteolysis and virus release. Previous studies have demonstrated that Vpu-induced degradation of CD4 occurs in the endoplasmic reticulum (ER), and that the proteolytic process is sequence specific requiring both the transmembrane and cytoplasmic domains of CD4. In the present study, we investigated the effects of Vpu expression on the intracellular membrane trafficking pathway of mammalian cells. In singly transfected cells, the HIV envelope glycoproteins and vesicular stomatitis virus glycoprotein (VSV G) were properly transported to the cell surface undergoing oligosaccharide modifications characteristic of their movement through the Golgi complex. In contrast, the cell surface delivery of glycoproteins was severely impeded in cells expressing Vpu. Biochemical analyses revealed that Vpu expression blocked the transfer of proteins from the ER-Golgi complex to the plasma membrane in a dose- and protein-dependent manner. Soluble gp120 exhibited extreme transport defects in the presence of Vpu, whereas transmembrane proteins (e.g., gp160, VSV) responded only moderately to wild-type Vpu. To gain insight into Vpu-mediated transport inhibition, we performed mutational analysis of the CK-2 phosphorylation sites (serines at 52 and 56) in the Vpu protein. CK-2 phosphorylation of Vpu has been shown to regulate the activity of the protein in reactions that involve the proteolysis of CD4 in the ER. We demonstrate here that the phosphorylation mutant is defective in both sequence-specific degradation of VRE-containing substrates and the transport inhibition of gp120 and VSV-G in the secretory pathway. Thus, these experiments have revealed that Vpu-mediated proteolysis and transport inhibition are mechanistically coupled requiring the same structural elements of the Vpu protein in both processes. We propose that the primary effect of Vpu expression is to impede the secretion process and then access glycoproteins bearing the VRE for Vpu-mediated proteolysis in the ER of mammalian cells.
D001483:Base Sequence; D001692:Biological Transport; D047390:Casein Kinase II; D002462:Cell Membrane / Q000378:metabolism*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1; D006367:HeLa Cells; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D047390:Casein Kinase II; D017931:DNA Primers; D015699:HIV Envelope Protein gp120; D054298:Human Immunodeficiency Virus Proteins; D008562:Membrane Glycoproteins; D017346:Protein Serine-Threonine Kinases; D014759:Viral Envelope Proteins; D054334:Viral Regulatory and Accessory Proteins; C028391:G protein, vesicular stomatitis virus; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins