Reviewed, UniProtKB/Swiss-Prot P03315 (POLS_SFV)

Names and origin

Protein names	Recommended name:     Structural polyprotein Alternative name(s):     p130 Cleaved into the following 6 chains:     1- Recommended name:             Capsid protein               EC=3.4.21.-         Alternative name(s):             Coat protein               Short name=C     2- Recommended name:             p62         Alternative name(s):             E3/E2     3- Recommended name:             E3 protein         Alternative name(s):             Spike glycoprotein E3     4- Recommended name:             E2 envelope glycoprotein         Alternative name(s):             Spike glycoprotein E2     5- Recommended name:             6K protein     6- Recommended name:             E1 envelope glycoprotein         Alternative name(s):             Spike glycoprotein E1
Organism	Semliki forest virus (SFV)
Taxonomic identifier	11033 [NCBI]
Taxonomic lineage	Viruses › ssRNA positive-strand viruses, no DNA stage › Togaviridae › Alphavirus › SFV complex
Virus host	Aedes [TaxID: 7158] Culex tritaeniorhynchus [TaxID: 7178] Atelerix albiventris (Middle-African hedgehog) [TaxID: 9368] Homo sapiens (Human) [TaxID: 9606] Rhipicephalus [TaxID: 34630] Quelea [TaxID: 158617] Halcyon [TaxID: 170865]

Protein attributes

Sequence length	1253 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is not processed.
Protein existence	Evidence at protein level.

General annotation (Comments)

Function	Capsid protein possesses a protease activity that results in its autocatalytic cleavage from the nascent structural protein. Following its self-cleavage, the capsid protein transiently associates with ribosomes, and within several minutes the protein binds to viral RNA and rapidly assembles into icosaedric core particles. The resulting nucleocapsid eventually associates with the cytoplasmic domain of E2 at the cell membrane, leading to budding and formation of mature virions. New virions attach to target cells, and after endocytosis their membrane fuses with the target cell membrane. This leads to the release of the nucleocapsid into the cytoplasm, followed by an uncoating event necessary for the genomic RNA to become accessible. The uncoating might be triggered by the interaction of capsid proteins with ribosomes. Binding of ribosomes would release the genomic RNA since the same region is genomic RNA-binding and ribosome-binding. E3 protein's function is unknown. E2 is responsible for viral attachment to target host cell, by binding to the cell receptor. Synthesized as a p62 precursor which is processed by furin at the cell membrane just before virion budding, giving rise to E2-E1 heterodimer. The p62-E1 heterodimer is stable, whereas E2-E1 is unstable and dissociate at low pH. p62 is processed at the last step, presumably to avoid E1 fusion activation before its final export to cell surface. E2 C-terminus contains a transitory transmembrane that would be disrupted by palmitoylation, resulting in reorientation of the C-terminal tail from lumenal to cytoplasmic side. This step is critical since E2 C-terminus is involved in budding by interacting with capsid proteins. This release of E2 C-terminus in cytoplasm occurs lately in protein export, and precludes premature assembly of particles at the endoplasmic reticulum membrane. 6K is a constitutive membrane protein involved in virus glycoprotein processing, membrane permeabilization, and the budding of viral particles. Present in low amount in virions, about 3% compared to viral glycoproteins. Because of its lipophilic properties, the 6K protein is postulated to influence the selection of lipids that interact with the transmembrane domains of the glycoproteins, which, in turn, affects the deformability of the bilayer required for the extreme curvature that occurs as budding proceeds. E1 is a class II viral fusion protein. Fusion activity is inactive as long as E1 is bound to E2 in mature virion. After virus attachment to target cell and endocytosis, acidification of the endosome would induce dissociation of E1/E2 heterodimer and concomitant trimerization of the E1 subunits. This E1 trimer is fusion active, and promotes release of viral nucleocapsid in cytoplasm after cell and viral membrane fusion. Efficient fusion requires the presence of cholesterol and sphingolipid in the target membrane. Fusion is optimal at levels of about 1 molecule of cholesterol per 2 molecules of phospholipids, and is specific for sterols containing a 3-beta-hydroxyl group.
Subunit structure	p62 and E1 form a heterodimer shortly after synthesis. Processing of p62 into E2 and E3 results in a heterodimer of E2 and E1. Spike at virion surface are constituted of three E2-E1 heterodimers. After target cell attachment and endocytosis, E1 change conformation to form homotrimers.
Subcellular location	Capsid protein: VirionBy similarity. CytoplasmBy similarity. p62: Virion membrane; Single-pass type I membrane proteinBy similarity. Cell membrane; Single-pass type I membrane proteinBy similarity. E2 envelope glycoprotein: Virion membrane; Single-pass type I membrane proteinBy similarity. Cell membrane; Single-pass type I membrane proteinBy similarity. E1 envelope glycoprotein: Virion membrane; Single-pass type I membrane proteinBy similarity. Cell membrane; Single-pass type I membrane proteinBy similarity. 6K protein: Cell membrane; Multi-pass membrane proteinBy similarity. Virion membrane; Multi-pass membrane proteinBy similarity.
Post-translational modification	Specific enzymatic cleavages in vivo yield mature proteins. Capsid protein is auto-cleaved during polyprotein translation, unmasking p62 signal peptide. The remaining polyprotein is then targeted to the endoplasmic reticulum, where host signal peptidase cleaves it into p62, 6K and E1 proteins. p62 is further processed to mature E3 and E2 by host furin in trans-Golgi vesicle. Protein processing process takes about 30 minutes at physiologic temperatures. The folding of the p62/6K/E1 precursor requires the formation of intrachain disulfide bonds and has been shown to involve a transient covalent interaction between the nascent and newly synthesized heterodimer and the host-cell chaperones, P4HB/PDI and PDIA3/ERp57. The folding pathway also includes non covalent interaction with human CANX/calnexin and CALR/calreticulin. Envelope E1, E2 and E3 proteins are N-glycosylated. E2 is palmitoylated via thioester bonds. These palmitoylations may induce disruption of the C-terminus transmembrane. This would result in the reorientation of E2 c-terminus from lumenal to cytoplasmic side. 6K protein is also palmitoylated with about four covalently bound fatty acids per molecule. E1 is stearoylated.
Miscellaneous	The mature virion nucleocapsid consists of 240 copies of the capsid protein. 80 spike trimers of E1 and E2 are present at the surface of mature virion. They project about 100 Angstroms from the outer surface and are located at the local and strict three fold axis of the icosaedral lattice. The glycoproteins splay out to form a protein shell or skirt covering most of the outer surface of the membrane bilayer. Structural polyprotein is translated from a subgenomic RNA synthesized during togavirus replication.
Sequence similarities	Contains 1 peptidase S3 domain.

Ontologies

Keywords
Cellular component	Capsid protein Cell membrane Cytoplasm Membrane Virion
Domain	Signal Transmembrane
Molecular function	Hydrolase Protease Serine protease
PTM	Cleavage on pair of basic residues Glycoprotein Lipoprotein Palmitate
Technical term	3D-structure Direct protein sequencing
Gene Ontology (GO)
Cellular component	virion membrane Inferred from electronic annotation. Source: UniProtKB-SubCell
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Molecule processing

Chain

1 – 267

267

Capsid protein

Chain

268 – 755

488

p62

Chain

268 – 333

66

E3 protein

Signal peptide

268 – 282

15

Not cleaved Potential

Chain

334 – 755

422

E2 envelope glycoprotein

Chain

756 – 815

60

6K protein

Chain

816 – 1253

438

E1 envelope glycoprotein

Regions

Topological domain

268 – 701

434

Extracellular Potential

Transmembrane

702 – 722

21

Potential

Topological domain

723 – 755

33

Cytoplasmic Potential

Topological domain

756 – 770

15

Extracellular Potential

Transmembrane

771 – 791

21

Potential

Topological domain

792

1

Cytoplasmic Potential

Transmembrane

793 – 813

21

Potential

Topological domain

814 – 1230

417

Extracellular Potential

Transmembrane

1231 – 1251

21

Potential

Topological domain

1252 – 1253

2

Cytoplasmic Potential

Domain

112 – 267

156

Peptidase S3

Region

1 – 113

113

Intrinsically disordered, in contact with genomic RNA in nucleocapsid Potential

Region

94 – 106

13

Ribosome-binding

Region

728 – 748

21

Transient transmembrane before p62-6K protein processing Potential

Region

899 – 916

18

E1 fusion peptide loop

Sites

Active site

145

1

Charge relay system By similarity

Active site

151

1

Charge relay system By similarity

Active site

219

1

Charge relay system By similarity

Site

267 – 268

2

Cleavage; by capsid protein

Site

333 – 334

2

Cleavage; by host furin

Site

755 – 756

2

Cleavage; by host signal peptidase

Site

815 – 816

2

Cleavage; by host signal peptidase

Amino acid modifications

Lipidation

718

1

S-palmitoyl cysteine; by host Potential

Lipidation

728

1

S-palmitoyl cysteine; by host By similarity

Lipidation

748

1

S-palmitoyl cysteine; by host By similarity

Lipidation

749

1

S-palmitoyl cysteine; by host By similarity

Lipidation

1248

1

S-stearoyl cysteine; by host

Glycosylation

280

1

N-linked (GlcNAc...) Potential

Glycosylation

327

1

N-linked (GlcNAc...) Potential

Glycosylation

533

1

N-linked (GlcNAc...) Potential

Glycosylation

595

1

N-linked (GlcNAc...) Potential

Glycosylation

956

1

N-linked (GlcNAc...)

Disulfide bond

119 ↔ 134

Disulfide bond

864 ↔ 929

Disulfide bond

877 ↔ 909

Disulfide bond

878 ↔ 911

Disulfide bond

883 ↔ 893

Disulfide bond

1074 ↔ 1086

Disulfide bond

1116 ↔ 1191

Disulfide bond

1121 ↔ 1195

Disulfide bond

1143 ↔ 1185

Experimental info

Mutagenesis

267

1

W → A or R: Complete loss of cleavage by capsid protease

Mutagenesis

330

1

R → S: Complete loss of p62 precursor processing

Mutagenesis

333

1

R → F: Complete loss of p62 precursor processing

Mutagenesis

755

1

A → F: Complete loss of p62 precursor-6K cleavage

Mutagenesis

815

1

A → F: Complete loss of 6K protein-E1 envelope glycoprotein cleavage

Mutagenesis

859

1

L → F: E1 fusion is less cholesterol and sphingolipid dependent

Mutagenesis

890

1

D → A: Shifts the pH threshold for fusion to a more acidic range

Mutagenesis

894

1

K → Q: No effect on E1 fusion activity

Mutagenesis

898

1

G → A: Shifts the pH threshold for fusion to a more acidic range

Mutagenesis

898

1

G → D: No effect on E1 fusion activity

Mutagenesis

901

1

P → D: Retention of E1 protein in endoplasmic reticulum

Mutagenesis

903

1

M → L: No effect on E1 fusion activity

Mutagenesis

906

1

G → A: Shifts the pH threshold for fusion to a more acidic range

Mutagenesis

906

1

G → D: Complete loss of E1 fusion activity

Mutagenesis

906

1

G → P: Retention of E1 protein in endoplasmic reticulum

Mutagenesis

993

1

V → A: E1 fusion is less cholesterol and sphingolipid dependent

Secondary structure

1

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1253

Helix Strand Turn

Details...

Beta strand

120 – 125

6

Beta strand

128 – 134

7

Beta strand

139 – 141

3

Beta strand

148 – 152

5

Helix

153 – 156

4

Beta strand

161 – 163

3

Turn

164 – 167

4

Beta strand

168 – 172

5

Helix

175 – 180

6

Beta strand

190 – 195

6

Beta strand

198 – 203

6

Beta strand

206 – 210

5

Beta strand

222 – 224

3

Beta strand

230 – 239

10

Beta strand

241 – 251

11

Beta strand

253 – 259

7

Sequences

	Sequence		Length	Mass (Da)
	P03315-1 [UniParc]. Last modified July 21, 1986. Version 1. Checksum: 2A73228D08B82AC5	FASTA	1,253	138,017
	10 20 30 40 50 60 MNYIPTQTFY GRRWRPRPAA RPWPLQATPV APVVPDFQAQ QMQQLISAVN ALTMRQNAIA 70 80 90 100 110 120 PARPPKPKKK KTTKPKPKTQ PKKINGKTQQ QKKKDKQADK KKKKPGKRER MCMKIENDCI 130 140 150 160 170 180 FEVKHEGKVT GYACLVGDKV MKPAHVKGVI DNADLAKLAF KKSSKYDLEC AQIPVHMRSD 190 200 210 220 230 240 ASKYTHEKPE GHYNWHHGAV QYSGGRFTIP TGAGKPGDSG RPIFDNKGRV VAIVLGGANE 250 260 270 280 290 300 GSRTALSVVT WNKDMVTRVT PEGSEEWSAP LITAMCVLAN ATFPCFQPPC VPCCYENNAE 310 320 330 340 350 360 ATLRMLEDNV DRPGYYDLLQ AALTCRNGTR HRRSVSQHFN VYKATRPYIA YCADCGAGHS 370 380 390 400 410 420 CHSPVAIEAV RSEATDGMLK IQFSAQIGID KSDNHDYTKI RYADGHAIEN AVRSSLKVAT 430 440 450 460 470 480 SGDCFVHGTM GHFILAKCPP GEFLQVSIQD TRNAVRACRI QYHHDPQPVG REKFTIRPHY 490 500 510 520 530 540 GKEIPCTTYQ QTTAETVEEI DMHMPPDTPD RTLLSQQSGN VKITVGGKKV KYNCTCGTGN 550 560 570 580 590 600 VGTTNSDMTI NTCLIEQCHV SVTDHKKWQF NSPFVPRADE PARKGKVHIP FPLDNITCRV 610 620