Abstract
The assembly and budding of Sindbis virus, a prototypic member of the alphavirus subgroup in the family Togaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embedded glycoproteins E1 and E2. These glycoproteins are modified posttranslationally by the addition of palmitic acid, and inhibitors of acylation interfere with this budding process (M.J. Schlesinger and C. Malfer, J. Biol. Chem. 257:9887-9890, 1982). This report describes the use of site-directed mutagenesis to identify two of the acylation sites in the E2 glycoprotein as the cysteines near the carboxyl terminus of the protein which is oriented to the cytoplasmic domain of this type 1 transmembrane protein. Additional mutations were made at two prolines within a hydrophobic sequence of E2 that is highly conserved among several alphaviruses, and the mutant viruses were aberrant in assembly and particle formation. These data support earlier studies indicating that the native structure of the cytoplasmic domain of E2 is essential for proper assembly of this enveloped virus.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aliperti G., Schlesinger M. J. Evidence for an autoprotease activity of sindbis virus capsid protein. Virology. 1978 Oct 15;90(2):366–369. doi: 10.1016/0042-6822(78)90321-5. [DOI] [PubMed] [Google Scholar]
- Dalgarno L., Rice C. M., Strauss J. H. Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins. Virology. 1983 Aug;129(1):170–187. doi: 10.1016/0042-6822(83)90404-x. [DOI] [PubMed] [Google Scholar]
- Gaedigk-Nitschko K., Ding M. X., Levy M. A., Schlesinger M. J. Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure. Virology. 1990 Mar;175(1):282–291. doi: 10.1016/0042-6822(90)90210-i. [DOI] [PubMed] [Google Scholar]
- Gaedigk-Nitschko K., Schlesinger M. J. Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding. Virology. 1991 Jul;183(1):206–214. doi: 10.1016/0042-6822(91)90133-v. [DOI] [PubMed] [Google Scholar]
- Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
- Hahn C. S., Lustig S., Strauss E. G., Strauss J. H. Western equine encephalitis virus is a recombinant virus. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5997–6001. doi: 10.1073/pnas.85.16.5997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hahn C. S., Strauss E. G., Strauss J. H. Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4648–4652. doi: 10.1073/pnas.82.14.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kinney R. M., Johnson B. J., Brown V. L., Trent D. W. Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins. Virology. 1986 Jul 30;152(2):400–413. doi: 10.1016/0042-6822(86)90142-x. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Levinson R. S., Strauss J. H., Strauss E. G. Complete sequence of the genomic RNA of O'nyong-nyong virus and its use in the construction of alphavirus phylogenetic trees. Virology. 1990 Mar;175(1):110–123. doi: 10.1016/0042-6822(90)90191-s. [DOI] [PubMed] [Google Scholar]
- Liljeström P., Garoff H. Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J Virol. 1991 Jan;65(1):147–154. doi: 10.1128/jvi.65.1.147-154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magee A. I., Koyama A. H., Malfer C., Wen D., Schlesinger M. J. Release of fatty acids from virus glycoproteins by hydroxylamine. Biochim Biophys Acta. 1984 Apr 10;798(2):156–166. doi: 10.1016/0304-4165(84)90298-8. [DOI] [PubMed] [Google Scholar]
- Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
- Rice C. M., Bell J. R., Hunkapiller M. W., Strauss E. G., Strauss J. H. Isolation and characterization of the hydrophobic COOH-terminal domains of the sindbis virion glycoproteins. J Mol Biol. 1982 Jan 15;154(2):355–378. doi: 10.1016/0022-2836(82)90069-9. [DOI] [PubMed] [Google Scholar]
- Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [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]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlesinger M. J., Malfer C. Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. J Biol Chem. 1982 Sep 10;257(17):9887–9890. [PubMed] [Google Scholar]
- Schmidt M. F., Bracha M., Schlesinger M. J. Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1687–1691. doi: 10.1073/pnas.76.4.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]