PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
 
J Virol. 1996 November; 70(11): 8029–8040.
PMCID: PMC190876

Mutations in the carboxyl-terminal hydrophobic sequence of human cytomegalovirus glycoprotein B alter transport and protein chaperone binding.

Abstract

Human cytomegalovirus glycoprotein B (gB) plays a role in the fusion of the virion envelope with the host cell membrane and in syncytium formation in infected cells. Hydrophobic sequences at the carboxyl terminus, amino acids (aa) 714 to 771, anchor gB in the lipid bilayer, but the unusual length of this domain suggests that it may serve another role in gB structure. To explore the function(s) of this region, we deleted aa 717 to 747 (gB deltaI mutation), aa 751 to 771 (gB deltaII mutation), and aa 717 to 772 (gB deltaI-II mutation) and constructed a substitution mutation, Lys-748 to Val (Lys748Val)-Asn749Ala-Pro750Ile (gB KNPm). Mutated forms of gB were expressed in U373 glioblastoma cells and subjected to analysis by flow cytometry, confocal microscopy, and immunoprecipitation. Mutations gB deltaI-II and gB deltaII alone caused secretion of gB into the medium, confirming that aa 751 to 771 function as a membrane anchor. In contrast, mutations gB deltaI and gB KNPm blocked cell surface expression and arrested gB transport in the endoplasmic reticulum (ER). Detailed examination of gB deltaI and gB KNPm with a panel of monoclonal antibodies showed that the mutated forms were indistinguishable from wild-type gB in conformation and formed oligomers; however, they remained sensitive to endoglycosidase H and did not undergo endoproteolytic cleavage. Analysis of protein complexes formed by gB and molecular chaperones in the ER showed that calnexin and calreticulin, lectin-like chaperones, bound equal amounts of uncleaved wild-type gB, gB deltaI, and gB KNPm, but the glucose-regulated proteins 78 (BiP) and 94 formed stable complexes only with the mutated forms, causing their retention in the ER. Our studies show that aa 714 to 750 are key residues in the architecture of gB molecules and that the ER chaperones, which facilitate gB folding and monitor the quality of glycoproteins, detect subtle changes in folding intermediates that are conferred by mutations in this region.

Full Text

The Full Text of this article is available as a PDF (674K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bergeron JJ, Brenner MB, Thomas DY, Williams DB. Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci. 1994 Mar;19(3):124–128. [PubMed]
  • Billstrom MA, Britt WJ. Postoligomerization folding of human cytomegalovirus glycoprotein B: identification of folding intermediates and importance of disulfide bonding. J Virol. 1995 Nov;69(11):7015–7022. [PMC free article] [PubMed]
  • Bonifacino JS, Lippincott-Schwartz J. Degradation of proteins within the endoplasmic reticulum. Curr Opin Cell Biol. 1991 Aug;3(4):592–600. [PubMed]
  • Boppana SB, Polis MA, Kramer AA, Britt WJ, Koenig S. Virus-specific antibody responses to human cytomegalovirus (HCMV) in human immunodeficiency virus type 1-infected persons with HCMV retinitis. J Infect Dis. 1995 Jan;171(1):182–185. [PubMed]
  • Britt WJ, Vugler LG. Processing of the gp55-116 envelope glycoprotein complex (gB) of human cytomegalovirus. J Virol. 1989 Jan;63(1):403–410. [PMC free article] [PubMed]
  • Britt WJ, Vugler LG. Antiviral antibody responses in mothers and their newborn infants with clinical and subclinical congenital cytomegalovirus infections. J Infect Dis. 1990 Feb;161(2):214–219. [PubMed]
  • Britt WJ, Vugler LG. Oligomerization of the human cytomegalovirus major envelope glycoprotein complex gB (gp55-116). J Virol. 1992 Nov;66(11):6747–6754. [PMC free article] [PubMed]
  • Chee MS, Bankier AT, Beck S, Bohni R, Brown CM, Cerny R, Horsnell T, Hutchison CA, 3rd, Kouzarides T, Martignetti JA, et al. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. [PubMed]
  • Chen W, Helenius J, Braakman I, Helenius A. Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6229–6233. [PubMed]
  • Cranage MP, Kouzarides T, Bankier AT, Satchwell S, Weston K, Tomlinson P, Barrell B, Hart H, Bell SE, Minson AC, et al. Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus. EMBO J. 1986 Nov;5(11):3057–3063. [PubMed]
  • Creighton TE. An empirical approach to protein conformation stability and flexibility. Biopolymers. 1983 Jan;22(1):49–58. [PubMed]
  • David V, Hochstenbach F, Rajagopalan S, Brenner MB. Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J Biol Chem. 1993 May 5;268(13):9585–9592. [PubMed]
  • Drew WL. Cytomegalovirus infection in patients with AIDS. J Infect Dis. 1988 Aug;158(2):449–456. [PubMed]
  • Drew WL, Sweet ES, Miner RC, Mocarski ES. Multiple infections by cytomegalovirus in patients with acquired immunodeficiency syndrome: documentation by Southern blot hybridization. J Infect Dis. 1984 Dec;150(6):952–953. [PubMed]
  • Freeman WR, Henderly DE, Lipson BK, Rao NA, Levine AM. Retinopathy before the diagnosis of AIDS. Ann Ophthalmol. 1989 Dec;21(12):468–474. [PubMed]
  • Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. [PubMed]
  • Hammond C, Braakman I, Helenius A. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):913–917. [PubMed]
  • Hammond C, Helenius A. Folding of VSV G protein: sequential interaction with BiP and calnexin. Science. 1994 Oct 21;266(5184):456–458. [PubMed]
  • Hammond C, Helenius A. Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus. J Cell Biol. 1994 Jul;126(1):41–52. [PMC free article] [PubMed]
  • Helenius A, Marquardt T, Braakman I. The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol. 1992 Aug;2(8):227–231. [PubMed]
  • Jackson MR, Cohen-Doyle MF, Peterson PA, Williams DB. Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90). Science. 1994 Jan 21;263(5145):384–387. [PubMed]
  • Kim PS, Arvan P. Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum. J Cell Biol. 1995 Jan;128(1-2):29–38. [PMC free article] [PubMed]
  • Klausner RD, Sitia R. Protein degradation in the endoplasmic reticulum. Cell. 1990 Aug 24;62(4):611–614. [PubMed]
  • Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. [PubMed]
  • Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PubMed]
  • Moss B, Elroy-Stein O, Mizukami T, Alexander WA, Fuerst TR. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. [PubMed]
  • Nauseef WM, McCormick SJ, Clark RA. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. J Biol Chem. 1995 Mar 3;270(9):4741–4747. [PubMed]
  • Navarro D, Paz P, Tugizov S, Topp K, La Vail J, Pereira L. Glycoprotein B of human cytomegalovirus promotes virion penetration into cells, transmission of infection from cell to cell, and fusion of infected cells. Virology. 1993 Nov;197(1):143–158. [PubMed]
  • Navarro D, Qadri I, Pereira L. A mutation in the ectodomain of herpes simplex virus 1 glycoprotein B causes defective processing and retention in the endoplasmic reticulum. Virology. 1991 Sep;184(1):253–264. [PubMed]
  • Otteken A, Moss B. Calreticulin interacts with newly synthesized human immunodeficiency virus type 1 envelope glycoprotein, suggesting a chaperone function similar to that of calnexin. J Biol Chem. 1996 Jan 5;271(1):97–103. [PubMed]
  • Ou WJ, Cameron PH, Thomas DY, Bergeron JJ. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature. 1993 Aug 26;364(6440):771–776. [PubMed]
  • Pass RF, August AM, Dworsky M, Reynolds DW. Cytomegalovirus infection in day-care center. N Engl J Med. 1982 Aug 19;307(8):477–479. [PubMed]
  • Pass RF, Stagno S, Myers GJ, Alford CA. Outcome of symptomatic congenital cytomegalovirus infection: results of long-term longitudinal follow-up. Pediatrics. 1980 Nov;66(5):758–762. [PubMed]
  • Pellett PE, Kousoulas KG, Pereira L, Roizman B. Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants. J Virol. 1985 Jan;53(1):243–253. [PMC free article] [PubMed]
  • Pereira L. Function of glycoprotein B homologues of the family herpesviridae. Infect Agents Dis. 1994 Feb;3(1):9–28. [PubMed]
  • Pereira L, Hoffman M, Tatsuno M, Dondero D. Polymorphism of human cytomegalovirus glycoproteins characterized by monoclonal antibodies. Virology. 1984 Nov;139(1):73–86. [PubMed]
  • Pereira L, Stagno S, Hoffman M, Volanakis JE. Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections. Infect Immun. 1983 Jan;39(1):100–108. [PMC free article] [PubMed]
  • Peterson JR, Ora A, Van PN, Helenius A. Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol Biol Cell. 1995 Sep;6(9):1173–1184. [PMC free article] [PubMed]
  • Qadri I, Gimeno C, Navarro D, Pereira L. Mutations in conformation-dependent domains of herpes simplex virus 1 glycoprotein B affect the antigenic properties, dimerization, and transport of the molecule. Virology. 1991 Jan;180(1):135–152. [PubMed]
  • Qadri I, Navarro D, Paz P, Pereira L. Assembly of conformation-dependent neutralizing domains on glycoprotein B of human cytomegalovirus. J Gen Virol. 1992 Nov;73(Pt 11):2913–2921. [PubMed]
  • Rajagopalan S, Brenner MB. Calnexin retains unassembled major histocompatibility complex class I free heavy chains in the endoplasmic reticulum. J Exp Med. 1994 Jul 1;180(1):407–412. [PMC free article] [PubMed]
  • Ramakrishnan M, Tugizov S, Pereira L, Lee AS. Conformation-defective herpes simplex virus 1 glycoprotein B activates the promoter of the grp94 gene that codes for the 94-kD stress protein in the endoplasmic reticulum. DNA Cell Biol. 1995 May;14(5):373–384. [PubMed]
  • Rasile L, Ghosh K, Raviprakash K, Ghosh HP. Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring. J Virol. 1993 Aug;67(8):4856–4866. [PMC free article] [PubMed]
  • Rasmussen L, Nelson M, Neff M, Merigan TC., Jr Characterization of two different human cytomegalovirus glycoproteins which are targets for virus neutralizing antibody. Virology. 1988 Apr;163(2):308–318. [PubMed]
  • Reschke M, Reis B, Nöding K, Rohsiepe D, Richter A, Mockenhaupt T, Garten W, Radsak K. Constitutive expression of human cytomegalovirus glycoprotein B (gpUL55) with mutagenized carboxy-terminal hydrophobic domains. J Gen Virol. 1995 Jan;76(Pt 1):113–122. [PubMed]
  • Romagnoli P, Germain RN. Inhibition of invariant chain (Ii)-calnexin interaction results in enhanced degradation of Ii but does not prevent the assembly of alpha beta Ii complexes. J Exp Med. 1995 Dec 1;182(6):2027–2036. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Spaete RR, Saxena A, Scott PI, Song GJ, Probert WS, Britt WJ, Gibson W, Rasmussen L, Pachl C. Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. J Virol. 1990 Jun;64(6):2922–2931. [PMC free article] [PubMed]
  • Spaete RR, Thayer RM, Probert WS, Masiarz FR, Chamberlain SH, Rasmussen L, Merigan TC, Pachl C. Human cytomegalovirus strain Towne glycoprotein B is processed by proteolytic cleavage. Virology. 1988 Nov;167(1):207–225. [PubMed]
  • Stagno S, Pass RF, Dworsky ME, Henderson RE, Moore EG, Walton PD, Alford CA. Congenital cytomegalovirus infection: The relative importance of primary and recurrent maternal infection. N Engl J Med. 1982 Apr 22;306(16):945–949. [PubMed]
  • Tarentino AL, Gómez CM, Plummer TH., Jr Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry. 1985 Aug 13;24(17):4665–4671. [PubMed]
  • Tatu U, Hammond C, Helenius A. Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment. EMBO J. 1995 Apr 3;14(7):1340–1348. [PubMed]
  • Tector M, Salter RD. Calnexin influences folding of human class I histocompatibility proteins but not their assembly with beta 2-microglobulin. J Biol Chem. 1995 Aug 18;270(33):19638–19642. [PubMed]
  • Tugizov S, Navarro D, Paz P, Wang Y, Qadri I, Pereira L. Function of human cytomegalovirus glycoprotein B: syncytium formation in cells constitutively expressing gB is blocked by virus-neutralizing antibodies. Virology. 1994 Jun;201(2):263–276. [PubMed]
  • Tugizov S, Wang Y, Qadri I, Navarro D, Maidji E, Pereira L. Mutated forms of human cytomegalovirus glycoprotein B are impaired in inducing syncytium formation. Virology. 1995 Jun 1;209(2):580–591. [PubMed]
  • Vey M, Schäfer W, Reis B, Ohuchi R, Britt W, Garten W, Klenk HD, Radsak K. Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin. Virology. 1995 Jan 10;206(1):746–749. [PubMed]
  • Wada I, Imai S, Kai M, Sakane F, Kanoh H. Chaperone function of calreticulin when expressed in the endoplasmic reticulum as the membrane-anchored and soluble forms. J Biol Chem. 1995 Sep 1;270(35):20298–20304. [PubMed]
  • Ward CL, Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995 Oct 6;83(1):121–127. [PubMed]
  • Ware FE, Vassilakos A, Peterson PA, Jackson MR, Lehrman MA, Williams DB. The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem. 1995 Mar 3;270(9):4697–4704. [PubMed]
  • Weiss CD, White JM. Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein. J Virol. 1993 Dec;67(12):7060–7066. [PMC free article] [PubMed]
  • Wooden SK, Li LJ, Navarro D, Qadri I, Pereira L, Lee AS. Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I. Mol Cell Biol. 1991 Nov;11(11):5612–5623. [PMC free article] [PubMed]
  • Yamashita Y, Shimokata K, Mizuno S, Daikoku T, Tsurumi T, Nishiyama Y. Calnexin acts as a molecular chaperone during the folding of glycoprotein B of human cytomegalovirus. J Virol. 1996 Apr;70(4):2237–2246. [PMC free article] [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)