Amino-terminal sequence analysis of the structural proteins of Sindbis virus.

Abstract

The structural proteins of Sindbis virus, an enveloped virus which belongs to the Togavirus family, have been subjected to automated Edman degradation using improved techniques. Extensive NHrterminal sequences of about 50 residues were determined for each of the two membrane glycoproteins. In both cases the NH2 terminus of the molecule was found to be similar in composition to typical water-soluble proteins. The viral capsid protein was found to have a blocked a-amino group. This is consistent with other observations that viral proteins derived from the NH2 terminus of precursor molecules are often blocked.Sindbis virus is a simple enveloped virus which matures by budding through a modified host cell plasma membrane (1). The virus particle contains only three structural proteins (2). One of these, the capsid protein, can be isolated from virus particles in the form of a nucleocapsid structure also containing the viral RNA (3). In the intact virus particle, this nucleocapsid is surrounded by a lipid envelope, largely in the form of a bilayer (4). Associated with the envelope are the other two structural proteins, glycoproteins El and E2. Although most of their mass is external to the lipid bilayer, they have the properties of integral membrane proteins. Mild detergent treatment is required to separate El and E2 from the other components of the virus (3,5), and at least one is a transmembrane protein because it extends completely through the bilayer and can be crosslinked to the capsid protein by suitable reagents (6).It has been shown that all three Sindbis structural proteins as well as those of the closely related Semliki Forest virus are formed by cleavage of a single precursor polypeptide (7-9), probably while the polypeptide chain is still nascent. The capsid protein is released into the cytoplasm of the cell; in contrast, El and E2 are inserted into the lumen of the rough endoplasmic reticulum (10). At some time after synthesis, they appear glycosylated at the cell plasmalemma, where they diffuse freely over the surface of the cell (11). The budding process appears to involve an interaction between preformed nucleocapsids and that region of one or both of the envelope proteins exposed at the cytoplasmic face of the plasmalemma. This MATERIALS AND METHODS Protein Preparation. The growth and purification of Sindbis virus and the separation of the structural proteins will be described in detail elsewhere. Briefly, the HR strain of Sindbis virus, grown at low multiplicity of infection in primary chicken embryo fibroblasts, was concentrated from culture fluid by precipitation with polyethylene glycol followed by pelleting onto a fluorocarbon cushion. The resuspended virus was then purified by sucrose gradient velocity centrifugation followed by isopycnic centrifugation in sucrose/2H20 gradients. These steps of the procedure were similar to those described previously (14). After disruption with Triton X-100, capsids were removed by centrifugation and the two envelope glycoproteins were separated by pas...

Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins.

“…The identification of the NH2 terminus and the COOH terminus of each protein is discussed below. The deduced amino acid sequence is in precise agreement with the NH2-terminal sequences found by automated sequence analysis of El and E2 (6), of PE2 (the precursor to E3 and E2) J. R. Bell and M. W. Hunkapiller, personal communication), and of the hydrophobic "roots" of El and E2 derived by treatment of intact virions with a-chymotrypsin (unpublished data). In addition, sequences of many of the tryptic peptides from the Sindbis (strain Sa-AR-86) capsid protein have been published recently (7); most ofthese sequence data are consistent with our deduced sequence for the capsid protein.…”

Alphavirus Replication: The Role of Cardiac Glycosides and Ion Concentration in Host Cells

“…For example, increasing the NaCl concentration in tissue culture medium directly inhibits maturation and release of the Sindbis virus, Semliki Forest, and vesicular Stomatitis virus [17]. By contrast, elevated NaCl concentrations were also associated with increased transcription efficiency of Sindbis virus messenger RNA (mRNA) [18]. The significance of the Na + ion concentration and its impact on reducing viral yield was also considered in experiments focused on Chikungunya virus (CHIKV) infection in human osteosarcoma cells.…”

Three Genes Code for Rubella Virus Structural Proteins E1, E2a, E2b and C

“…Many viral proteins are known to have blocked amino termini, as shown, e.g. for Mengo virus (Ziola & Scraba, 1976), Sindbis virus (Bell et al, 1978 ;Boege et al, 1980), Semliki Forest virus (Boege et al, 1980;Kalkkinen et al, 1980Kalkkinen et al, , 1981aKalkkinen et al, ) and adenovirus (J6rnvall et al, 1974. In almost all cases, when a blocked protein is formed by cleavage of a precursor, the blocked product is located at the amino terminus of the precursor polypeptide.…”