Abstract
A step in the maturation of Sindbis virus glycoproteins is the cleavage of the precursor glycoprotein PE2 into E3 and E2 by furin or a furin-like host cell protease. The results presented here suggest that PE2 cleavage is an obligatory event for Sindbis virus maturation in C6/36 cells and demonstrate that certain mutants display a cell-specific PE2 cleavage phenotype. We previously have described Sindbis virus variants which fail to cleave PE2 because of incorporation of a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage site but are viable in BHK-21 cells by virtue of an additional mutation at E2 216 or E2 191 (TRSB-NE2G216 and TRSB-NE2T191, respectively) (H. W. Heidner, K. L. McKnight, N. L. Davis, and R. E. Johnston, J. Virol. 68:2683-2692, 1994). Other viable PE2 cleavage-defective mutants were constructed by substituting the parental residue at E2 position 1 (Arg), with Leu or Val (TRSB-E2L1 and TRSB-E2V1, respectively) (H.W. Heidner and R. E. Johnston, J. Virol. 68:8064-8070, 1994). When grown in BHK-21 cells, all four of these viruses replicated normally and incorporated PE2 in place of E2 in released virions. However, growth of TRSB-NE2G216 and TRSB-NE2T191 was severely restricted in cultured arthropod cells (C6/36 cells). Analysis of infected C6/36 cells by flow cytometry demonstrated that the restricted growth of TRSB-NE2G216 and TRSB-NE2T191 was not due to an impaired ability to initiate infection. In addition, TRSB-NE2G216 and TRSB-NE2T191 remained growth restricted in C6/36 cells following introduction of in vitro transcriptions by electroporation. In contrast, the PE2 cleavage defect of TRSB-E2L1 and TRSB-E2V1 was cell type specific. In C6/36 cells, the majority of PE2 was converted to E2, and these viruses replicated normally in C6/36 cells. These results demonstrated a consistent link between expression of a PE2 cleavage defect and restricted growth in C6/36 cells and suggest that cleavage of PE2 is required for maturation of Sindbis virus late in infection of C6/36 cells.
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- Bauer K. D., Jacobberger J. W. Analysis of intracellular proteins. Methods Cell Biol. 1994;41:351–376. doi: 10.1016/s0091-679x(08)61728-8. [DOI] [PubMed] [Google Scholar]
- Davis N. L., Brown K. W., Greenwald G. F., Zajac A. J., Zacny V. L., Smith J. F., Johnston R. E. Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1. Virology. 1995 Sep 10;212(1):102–110. doi: 10.1006/viro.1995.1458. [DOI] [PubMed] [Google Scholar]
- De Bie I., Savaria D., Roebroek A. J., Day R., Lazure C., Van de Ven W. J., Seidah N. G. Processing specificity and biosynthesis of the Drosophila melanogaster convertases dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2. J Biol Chem. 1995 Jan 20;270(3):1020–1028. doi: 10.1074/jbc.270.3.1020. [DOI] [PubMed] [Google Scholar]
- Doms R. W., Lamb R. A., Rose J. K., Helenius A. Folding and assembly of viral membrane proteins. Virology. 1993 Apr;193(2):545–562. doi: 10.1006/viro.1993.1164. [DOI] [PubMed] [Google Scholar]
- Durbin R. K., Stollar V. A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2. Virology. 1984 Jun;135(2):331–344. doi: 10.1016/0042-6822(84)90190-9. [DOI] [PubMed] [Google Scholar]
- Erwin C., Brown D. T. Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants. J Virol. 1980 Dec;36(3):775–786. doi: 10.1128/jvi.36.3.775-786.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heidner H. W., Johnston R. E. The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice. J Virol. 1994 Dec;68(12):8064–8070. doi: 10.1128/jvi.68.12.8064-8070.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heidner H. W., McKnight K. L., Davis N. L., Johnston R. E. Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2. J Virol. 1994 Apr;68(4):2683–2692. doi: 10.1128/jvi.68.4.2683-2692.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hsieh P., Robbins P. W. Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells. J Biol Chem. 1984 Feb 25;259(4):2375–2382. [PubMed] [Google Scholar]
- Igarashi A. Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses. J Gen Virol. 1978 Sep;40(3):531–544. doi: 10.1099/0022-1317-40-3-531. [DOI] [PubMed] [Google Scholar]
- Jones K. J., Waite M. R., Bose H. R. Cleavage of a viral envelope precursor during the morphogenesis of Sindbis virus. J Virol. 1974 Apr;13(4):809–817. doi: 10.1128/jvi.13.4.809-817.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koblet H. The "merry-go-round": alphaviruses between vertebrate and invertebrate cells. Adv Virus Res. 1990;38:343–402. doi: 10.1016/s0065-3527(08)60866-0. [DOI] [PubMed] [Google Scholar]
- Kowal K. J., Stollar V. Temperature-sensitive host-dependent mutants of Sindbis virus. Virology. 1981 Oct 15;114(1):140–148. doi: 10.1016/0042-6822(81)90260-9. [DOI] [PubMed] [Google Scholar]
- Liljeström P., Lusa S., Huylebroeck D., Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol. 1991 Aug;65(8):4107–4113. doi: 10.1128/jvi.65.8.4107-4113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKnight K. L., Simpson D. A., Lin S. C., Knott T. A., Polo J. M., Pence D. F., Johannsen D. B., Heidner H. W., Davis N. L., Johnston R. E. Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes. J Virol. 1996 Mar;70(3):1981–1989. doi: 10.1128/jvi.70.3.1981-1989.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moehring J. M., Inocencio N. M., Robertson B. J., Moehring T. J. Expression of mouse furin in a Chinese hamster cell resistant to Pseudomonas exotoxin A and viruses complements the genetic lesion. J Biol Chem. 1993 Feb 5;268(4):2590–2594. [PubMed] [Google Scholar]
- Naim H. Y., Koblet H. The cleavage of p62, the precursor of E2 and E3, is an early and continuous event in Semliki Forest virus-infected Aedes albopictus cells. Arch Virol. 1990;110(3-4):221–237. doi: 10.1007/BF01311290. [DOI] [PubMed] [Google Scholar]
- Presely J. F., Brown D. T. The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus. J Virol. 1989 May;63(5):1975–1980. doi: 10.1128/jvi.63.5.1975-1980.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Presley J. F., Polo J. M., Johnston R. E., Brown D. T. Proteolytic processing of the Sindbis virus membrane protein precursor PE2 is nonessential for growth in vertebrate cells but is required for efficient growth in invertebrate cells. J Virol. 1991 Apr;65(4):1905–1909. doi: 10.1128/jvi.65.4.1905-1909.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Renz D., Brown D. T. Characteristics of Sindbis virus temperature-sensitive mutants in cultured BHK-21 and Aedes albopictus (Mosquito) cells. J Virol. 1976 Sep;19(3):775–781. doi: 10.1128/jvi.19.3.775-781.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rice C. M., Strauss J. H. Association of sindbis virion glycoproteins and their precursors. J Mol Biol. 1982 Jan 15;154(2):325–348. doi: 10.1016/0022-2836(82)90067-5. [DOI] [PubMed] [Google Scholar]
- Rice C. M., Strauss J. H. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2062–2066. doi: 10.1073/pnas.78.4.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roebroek A. J., Creemers J. W., Pauli I. G., Kurzik-Dumke U., Rentrop M., Gateff E. A., Leunissen J. A., Van de Ven W. J. Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif. J Biol Chem. 1992 Aug 25;267(24):17208–17215. [PubMed] [Google Scholar]
- Russell D. L., Dalrymple J. M., Johnston R. E. Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice. J Virol. 1989 Apr;63(4):1619–1629. doi: 10.1128/jvi.63.4.1619-1629.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Salminen A., Wahlberg J. M., Lobigs M., Liljeström P., Garoff H. Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry. J Cell Biol. 1992 Jan;116(2):349–357. doi: 10.1083/jcb.116.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlesinger M. J., Schlesinger S. Large-molecular-weight precursors of sindbis virus proteins. J Virol. 1973 Jun;11(6):1013–1016. doi: 10.1128/jvi.11.6.1013-1016.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith J. F., Brown D. T. Envelopments of Sindbis virus: synthesis and organization of proteins in cells infected with wild type and maturation-defective mutants. J Virol. 1977 Jun;22(3):662–678. doi: 10.1128/jvi.22.3.662-678.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wahlberg J. M., Boere W. A., Garoff H. The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation. J Virol. 1989 Dec;63(12):4991–4997. doi: 10.1128/jvi.63.12.4991-4997.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson D. G., Moehring J. M., Moehring T. J. A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2. J Virol. 1991 May;65(5):2332–2339. doi: 10.1128/jvi.65.5.2332-2339.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Curtis I., Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8052–8056. doi: 10.1073/pnas.85.21.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]