Functional Characterization of the Sindbis Virus E2 Glycoprotein by Transposon Linker-insertion Mutagenesis

Overview

Journal Virology

Specialty Microbiology

Date 2007 Feb 20

PMID 17306321

Citations 10

Authors

Chanakha K Navaratnarajah

Richard J Kuhn

Affiliations

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Abstract

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

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References

Mancini E, Clarke M, Gowen B, Rutten T, Fuller S . Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus. Mol Cell. 2000; 5(2):255-66. DOI: 10.1016/s1097-2765(00)80421-9. View

Paredes A, Heidner H, Prasad B, Johnston R, Chiu W . Structural localization of the E3 glycoprotein in attenuated Sindbis virus mutants. J Virol. 1998; 72(2):1534-41. PMC: 124635. DOI: 10.1128/JVI.72.2.1534-1541.1998. View

Pletnev S, Zhang W, Mukhopadhyay S, Fisher B, Hernandez R, Brown D . Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold. Cell. 2001; 105(1):127-136. PMC: 4140091. DOI: 10.1016/s0092-8674(01)00302-6. View

Lescar J, Roussel A, Wien M, Navaza J, Fuller S, WENGLER G . The Fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell. 2001; 105(1):137-48. DOI: 10.1016/s0092-8674(01)00303-8. View

Goryshin I, Reznikoff W . Tn5 in vitro transposition. J Biol Chem. 1998; 273(13):7367-74. DOI: 10.1074/jbc.273.13.7367. View

Sawai K, Meruelo D . Cell-specific transfection of choriocarcinoma cells by using Sindbis virus hCG expressing chimeric vector. Biochem Biophys Res Commun. 1998; 248(2):315-23. DOI: 10.1006/bbrc.1998.8922. View

Schlesinger S, Dubensky T . Alphavirus vectors for gene expression and vaccines. Curr Opin Biotechnol. 1999; 10(5):434-9. DOI: 10.1016/s0958-1669(99)00006-3. View

Hernandez R, Ferreira D, Sinodis C, Litton K, Brown D . Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity. J Virol. 2005; 79(12):7682-97. PMC: 1143637. DOI: 10.1128/JVI.79.12.7682-7697.2005. View

Prilusky J, Felder C, Zeev-Ben-Mordehai T, Rydberg E, Man O, Beckmann J . FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics. 2005; 21(16):3435-8. DOI: 10.1093/bioinformatics/bti537. View

10.

Chikkanna-Gowda C, Sheahan B, Fleeton M, Atkins G . Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12. Gene Ther. 2005; 12(16):1253-63. DOI: 10.1038/sj.gt.3302561. View

11.

Mukhopadhyay S, Zhang W, Gabler S, Chipman P, Strauss E, Strauss J . Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses. Structure. 2006; 14(1):63-73. PMC: 2757649. DOI: 10.1016/j.str.2005.07.025. View

12.

Zhang W, Mukhopadhyay S, Pletnev S, Baker T, Kuhn R, Rossmann M . Placement of the structural proteins in Sindbis virus. J Virol. 2002; 76(22):11645-58. PMC: 136788. DOI: 10.1128/jvi.76.22.11645-11658.2002. View

13.

Rost B, Yachdav G, Liu J . The PredictProtein server. Nucleic Acids Res. 2004; 32(Web Server issue):W321-6. PMC: 441515. DOI: 10.1093/nar/gkh377. View

14.

Herron P, Hughes G, Chandra G, Fielding S, Dyson P . Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA. Nucleic Acids Res. 2004; 32(14):e113. PMC: 514396. DOI: 10.1093/nar/gnh112. View

15.

von Bonsdorff C, Harrison S . Sindbis virus glycoproteins form a regular icosahedral surface lattice. J Virol. 1975; 16(1):141-5. PMC: 354642. DOI: 10.1128/JVI.16.1.141-145.1975. View

16.

Wirth D, Katz F, Small B, Lodish H . How a single Sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins. Cell. 1977; 10(2):253-63. DOI: 10.1016/0092-8674(77)90219-7. View

17.

von Bonsdorff C, Harrison S . Hexagonal glycoprotein arrays from Sindbis virus membranes. J Virol. 1978; 28(2):578-83. PMC: 354305. DOI: 10.1128/JVI.28.2.578-583.1978. View

18.

Schmidt M, Bracha M, SCHLESINGER M . Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Proc Natl Acad Sci U S A. 1979; 76(4):1687-91. PMC: 383455. DOI: 10.1073/pnas.76.4.1687. View

19.

Mayne J, Bell J, Strauss E, Strauss J . Pattern of glycosylation of Sindbis virus envelope proteins synthesized in hamster and chicken cells. Virology. 1985; 142(1):121-33. DOI: 10.1016/0042-6822(85)90427-1. View

20.

Melancon P, Garoff H . Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease. J Virol. 1987; 61(5):1301-9. PMC: 254103. DOI: 10.1128/JVI.61.5.1301-1309.1987. View