Abstract
The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.
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- Abdul Jabbar M., Nayak D. P. Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae. Mol Cell Biol. 1987 Apr;7(4):1476–1485. doi: 10.1128/mcb.7.4.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bergeron L., Sodroski J. Dissociation of unintegrated viral DNA accumulation from single-cell lysis induced by human immunodeficiency virus type 1. J Virol. 1992 Oct;66(10):5777–5787. doi: 10.1128/jvi.66.10.5777-5787.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bour S., Boulerice F., Wainberg M. A. Inhibition of gp160 and CD4 maturation in U937 cells after both defective and productive infections by human immunodeficiency virus type 1. J Virol. 1991 Dec;65(12):6387–6396. doi: 10.1128/jvi.65.12.6387-6396.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ciampor F., Bayley P. M., Nermut M. V., Hirst E. M., Sugrue R. J., Hay A. J. Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology. 1992 May;188(1):14–24. doi: 10.1016/0042-6822(92)90730-d. [DOI] [PubMed] [Google Scholar]
- Cohen E. A., Terwilliger E. F., Sodroski J. G., Haseltine W. A. Identification of a protein encoded by the vpu gene of HIV-1. Nature. 1988 Aug 11;334(6182):532–534. doi: 10.1038/334532a0. [DOI] [PubMed] [Google Scholar]
- Crise B., Buonocore L., Rose J. K. CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor. J Virol. 1990 Nov;64(11):5585–5593. doi: 10.1128/jvi.64.11.5585-5593.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cullen B. R. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol Rev. 1992 Sep;56(3):375–394. doi: 10.1128/mr.56.3.375-394.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dalgleish A. G., Beverley P. C., Clapham P. R., Crawford D. H., Greaves M. F., Weiss R. A. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature. 1984 Dec 20;312(5996):763–767. doi: 10.1038/312763a0. [DOI] [PubMed] [Google Scholar]
- Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Earl P. L., Moss B., Doms R. W. Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J Virol. 1991 Apr;65(4):2047–2055. doi: 10.1128/jvi.65.4.2047-2055.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuerst T. R., Earl P. L., Moss B. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol. 1987 Jul;7(7):2538–2544. doi: 10.1128/mcb.7.7.2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia J. V., Alfano J., Miller A. D. The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. J Virol. 1993 Mar;67(3):1511–1516. doi: 10.1128/jvi.67.3.1511-1516.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garcia J. V., Miller A. D. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991 Apr 11;350(6318):508–511. doi: 10.1038/350508a0. [DOI] [PubMed] [Google Scholar]
- Geyer H., Holschbach C., Hunsmann G., Schneider J. Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120. J Biol Chem. 1988 Aug 25;263(24):11760–11767. [PubMed] [Google Scholar]
- Gottesman S., Maurizi M. R. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56(4):592–621. doi: 10.1128/mr.56.4.592-621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grambas S., Bennett M. S., Hay A. J. Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Virology. 1992 Dec;191(2):541–549. doi: 10.1016/0042-6822(92)90229-i. [DOI] [PubMed] [Google Scholar]
- Haseltine W. A. Molecular biology of the human immunodeficiency virus type 1. FASEB J. 1991 Jul;5(10):2349–2360. doi: 10.1096/fasebj.5.10.1829694. [DOI] [PubMed] [Google Scholar]
- Helenius A. Unpacking the incoming influenza virus. Cell. 1992 May 15;69(4):577–578. doi: 10.1016/0092-8674(92)90219-3. [DOI] [PubMed] [Google Scholar]
- Helseth E., Olshevsky U., Gabuzda D., Ardman B., Haseltine W., Sodroski J. Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion. J Virol. 1990 Dec;64(12):6314–6318. doi: 10.1128/jvi.64.12.6314-6318.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holsinger L. J., Lamb R. A. Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology. 1991 Jul;183(1):32–43. doi: 10.1016/0042-6822(91)90115-r. [DOI] [PubMed] [Google Scholar]
- Hoxie J. A., Alpers J. D., Rackowski J. L., Huebner K., Haggarty B. S., Cedarbaum A. J., Reed J. C. Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV. Science. 1986 Nov 28;234(4780):1123–1127. doi: 10.1126/science.3095925. [DOI] [PubMed] [Google Scholar]
- Jabbar M. A., Nayak D. P. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane. J Virol. 1990 Dec;64(12):6297–6304. doi: 10.1128/jvi.64.12.6297-6304.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson M. R., Nilsson T., Peterson P. A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 1990 Oct;9(10):3153–3162. doi: 10.1002/j.1460-2075.1990.tb07513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson M. R., Nilsson T., Peterson P. A. Retrieval of transmembrane proteins to the endoplasmic reticulum. J Cell Biol. 1993 Apr;121(2):317–333. doi: 10.1083/jcb.121.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kawamura I., Koga Y., Oh-Hori N., Onodera K., Kimura G., Nomoto K. Depletion of the surface CD4 molecule by the envelope protein of human immunodeficiency virus expressed in a human CD4+ monocytoid cell line. J Virol. 1989 Sep;63(9):3748–3754. doi: 10.1128/jvi.63.9.3748-3754.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klatzmann D., Champagne E., Chamaret S., Gruest J., Guetard D., Hercend T., Gluckman J. C., Montagnier L. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature. 1984 Dec 20;312(5996):767–768. doi: 10.1038/312767a0. [DOI] [PubMed] [Google Scholar]
- Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klausner R. D., Sitia R. Protein degradation in the endoplasmic reticulum. Cell. 1990 Aug 24;62(4):611–614. doi: 10.1016/0092-8674(90)90104-m. [DOI] [PubMed] [Google Scholar]
- Klimkait T., Strebel K., Hoggan M. D., Martin M. A., Orenstein J. M. The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release. J Virol. 1990 Feb;64(2):621–629. doi: 10.1128/jvi.64.2.621-629.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koga Y., Sasaki M., Yoshida H., Oh-Tsu M., Kimura G., Nomoto K. Disturbance of nuclear transport of proteins in CD4+ cells expressing gp160 of human immunodeficiency virus. J Virol. 1991 Oct;65(10):5609–5612. doi: 10.1128/jvi.65.10.5609-5612.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
- Kowalski M., Potz J., Basiripour L., Dorfman T., Goh W. C., Terwilliger E., Dayton A., Rosen C., Haseltine W., Sodroski J. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science. 1987 Sep 11;237(4820):1351–1355. doi: 10.1126/science.3629244. [DOI] [PubMed] [Google Scholar]
- Kundu A., Jabbar M. A., Nayak D. P. Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains. Mol Cell Biol. 1991 May;11(5):2675–2685. doi: 10.1128/mcb.11.5.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lamb R. A., Zebedee S. L., Richardson C. D. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell. 1985 Mar;40(3):627–633. doi: 10.1016/0092-8674(85)90211-9. [DOI] [PubMed] [Google Scholar]
- Leonard C. K., Spellman M. W., Riddle L., Harris R. J., Thomas J. N., Gregory T. J. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem. 1990 Jun 25;265(18):10373–10382. [PubMed] [Google Scholar]
- Lifson J. D., Reyes G. R., McGrath M. S., Stein B. S., Engleman E. G. AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen. Science. 1986 May 30;232(4754):1123–1127. doi: 10.1126/science.3010463. [DOI] [PubMed] [Google Scholar]
- Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
- Maddon P. J., Molineaux S. M., Maddon D. E., Zimmerman K. A., Godfrey M., Alt F. W., Chess L., Axel R. Structure and expression of the human and mouse T4 genes. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9155–9159. doi: 10.1073/pnas.84.24.9155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosek J. C., Reyes G. R., Weissman I. L. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988 Apr 8;53(1):55–67. doi: 10.1016/0092-8674(88)90487-4. [DOI] [PubMed] [Google Scholar]
- Nayak D. P., Jabbar M. A. Structural domains and organizational conformation involved in the sorting and transport of influenza virus transmembrane proteins. Annu Rev Microbiol. 1989;43:465–501. doi: 10.1146/annurev.mi.43.100189.002341. [DOI] [PubMed] [Google Scholar]
- Pinto L. H., Holsinger L. J., Lamb R. A. Influenza virus M2 protein has ion channel activity. Cell. 1992 May 1;69(3):517–528. doi: 10.1016/0092-8674(92)90452-i. [DOI] [PubMed] [Google Scholar]
- Rosenberg Z. F., Fauci A. S. Immunopathogenesis of HIV infection. FASEB J. 1991 Jul;5(10):2382–2390. doi: 10.1096/fasebj.5.10.1676689. [DOI] [PubMed] [Google Scholar]
- Sanchez-Pescador R., Power M. D., Barr P. J., Steimer K. S., Stempien M. M., Brown-Shimer S. L., Gee W. W., Renard A., Randolph A., Levy J. A. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science. 1985 Feb 1;227(4686):484–492. doi: 10.1126/science.2578227. [DOI] [PubMed] [Google Scholar]
- Schwartz S., Felber B. K., Fenyö E. M., Pavlakis G. N. Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J Virol. 1990 Nov;64(11):5448–5456. doi: 10.1128/jvi.64.11.5448-5456.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shaw A. S., Amrein K. E., Hammond C., Stern D. F., Sefton B. M., Rose J. K. The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain. Cell. 1989 Nov 17;59(4):627–636. doi: 10.1016/0092-8674(89)90008-1. [DOI] [PubMed] [Google Scholar]
- Shaw A. S., Chalupny J., Whitney J. A., Hammond C., Amrein K. E., Kavathas P., Sefton B. M., Rose J. K. Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol Cell Biol. 1990 May;10(5):1853–1862. doi: 10.1128/mcb.10.5.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shin J., Dunbrack R. L., Jr, Lee S., Strominger J. L. Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4. J Biol Chem. 1991 Jun 5;266(16):10658–10665. [PubMed] [Google Scholar]
- Shin J., Dunbrack R. L., Jr, Lee S., Strominger J. L. Signals for retention of transmembrane proteins in the endoplasmic reticulum studied with CD4 truncation mutants. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1918–1922. doi: 10.1073/pnas.88.5.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shin J., Lee S., Strominger J. L. Translocation of TCR alpha chains into the lumen of the endoplasmic reticulum and their degradation. Science. 1993 Mar 26;259(5103):1901–1904. doi: 10.1126/science.8456316. [DOI] [PubMed] [Google Scholar]
- Sodroski J., Goh W. C., Rosen C., Campbell K., Haseltine W. A. Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. 1986 Jul 31-Aug 6Nature. 322(6078):470–474. doi: 10.1038/322470a0. [DOI] [PubMed] [Google Scholar]
- Somasundaran M., Robinson H. L. A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion. J Virol. 1987 Oct;61(10):3114–3119. doi: 10.1128/jvi.61.10.3114-3119.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stevenson M., Bukrinsky M., Haggerty S. HIV-1 replication and potential targets for intervention. AIDS Res Hum Retroviruses. 1992 Feb;8(2):107–117. doi: 10.1089/aid.1992.8.107. [DOI] [PubMed] [Google Scholar]
- Stevenson M., Meier C., Mann A. M., Chapman N., Wasiak A. Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for persistence in AIDS. Cell. 1988 May 6;53(3):483–496. doi: 10.1016/0092-8674(88)90168-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strebel K., Klimkait T., Maldarelli F., Martin M. A. Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein. J Virol. 1989 Sep;63(9):3784–3791. doi: 10.1128/jvi.63.9.3784-3791.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strebel K., Klimkait T., Martin M. A. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science. 1988 Sep 2;241(4870):1221–1223. doi: 10.1126/science.3261888. [DOI] [PubMed] [Google Scholar]
- Sugrue R. J., Bahadur G., Zambon M. C., Hall-Smith M., Douglas A. R., Hay A. J. Specific structural alteration of the influenza haemagglutinin by amantadine. EMBO J. 1990 Nov;9(11):3469–3476. doi: 10.1002/j.1460-2075.1990.tb07555.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sugrue R. J., Hay A. J. Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology. 1991 Feb;180(2):617–624. doi: 10.1016/0042-6822(91)90075-M. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terwilliger E. F., Cohen E. A., Lu Y. C., Sodroski J. G., Haseltine W. A. Functional role of human immunodeficiency virus type 1 vpu. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5163–5167. doi: 10.1073/pnas.86.13.5163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turner J. M., Brodsky M. H., Irving B. A., Levin S. D., Perlmutter R. M., Littman D. R. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 1990 Mar 9;60(5):755–765. doi: 10.1016/0092-8674(90)90090-2. [DOI] [PubMed] [Google Scholar]
- Willey R. L., Bonifacino J. S., Potts B. J., Martin M. A., Klausner R. D. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9580–9584. doi: 10.1073/pnas.85.24.9580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Willey R. L., Maldarelli F., Martin M. A., Strebel K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992 Dec;66(12):7193–7200. doi: 10.1128/jvi.66.12.7193-7200.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Willey R. L., Maldarelli F., Martin M. A., Strebel K. Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes. J Virol. 1992 Jan;66(1):226–234. doi: 10.1128/jvi.66.1.226-234.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yao X. J., Göttlinger H., Haseltine W. A., Cohen E. A. Envelope glycoprotein and CD4 independence of vpu-facilitated human immunodeficiency virus type 1 capsid export. J Virol. 1992 Aug;66(8):5119–5126. doi: 10.1128/jvi.66.8.5119-5126.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]