P13896 (POLN_WEEV) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names	Recommended name: Non-structural polyprotein Alternative name(s): Polyprotein nsP1234 Short name=P1234 Cleaved into the following 7 chains: P123 P123' mRNA-capping enzyme nsP1 EC=2.1.1.- EC=2.7.7.- Alternative name(s): Non-structural protein 1 Protease nsP2 EC=3.1.3.33 EC=3.4.22.- EC=3.6.1.15 EC=3.6.4.13 Alternative name(s): Non-structural protein 2 Short name=nsP2 Non-structural protein 3 Short name=nsP3 Non-structural protein 3' Short name=nsP3' RNA-directed RNA polymerase nsP4 EC=2.7.7.48 Alternative name(s): Non-structural protein 4 Short name=nsP4
Organism	Western equine encephalitis virus (WEEV) [Complete proteome]
Taxonomic identifier	11039 [NCBI]
Taxonomic lineage	Viruses › ssRNA positive-strand viruses, no DNA stage › Togaviridae › Alphavirus › WEEV complex
Virus host	Aedes [TaxID: 7158] Anopheles [TaxID: 7164] Culex tarsalis (Encephalitis mosquito) [TaxID: 7177] Rana pipiens (Northern leopard frog) [TaxID: 8404] Homo sapiens (Human) [TaxID: 9606] Carpodacus mexicanus (House finch) [TaxID: 30427] Thamnophis [TaxID: 34999] Spermophilus richardsonii (Richardson's ground squirrel) (Citellus richardsonii) [TaxID: 37591] Lepus americanus (Snowshoe hare) [TaxID: 48086] Passer domesticus (House sparrow) (Fringilla domestica) [TaxID: 48849]

Protein attributes

Sequence length	2466 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is further processed into a mature form.
Protein existence	Evidence at transcript level

General annotation (Comments)

Function	P123 and P123' are short-lived polyproteins, accumulating during early stage of infection. P123 is directly translated from the genome, whereas P123' is a product of the cleavage of P1234. They localize the viral replication complex to the cytoplasmic surface of modified endosomes and lysosomes. By interacting with nsP4, they start viral genome replication into antigenome. After these early events, P123 and P123' are cleaved sequentially into nsP1, nsP2 and nsP3/nsP3'. This sequence of delayed processing would allow correct assembly and membrane association of the RNA polymerase complex By similarity. nsP1 is a cytoplasmic capping enzyme. This function is necessary since all viral RNAs are synthesized in the cytoplasm, and host capping enzymes are restricted to the nucleus. The enzymatic reaction involves a covalent link between 7-methyl-GMP and nsP1, whereas eukaryotic capping enzymes form a covalent complex only with GMP. nsP1 capping would consist in the following reactions: GTP is first methylated and then forms the m7GMp-nsP1 complex, from which 7-methyl-GMP complex is transferred to the mRNA to create the cap structure. Palmitoylated nsP1 is remodeling host cell cytoskeleton, and induces filopodium-like structure formation at the surface of the host cell By similarity. nsP2 has two separate domain with different biological activities. The N-terminal section is part of the RNA polymerase complex and has RNA trisphosphatase and RNA helicase activity. The C-terminal section harbors a protease that specifically cleaves and releases the four mature proteins By similarity. nsP3 and nsP3' are essential for minus strand and subgenomic 26S mRNA synthesis By similarity. nsP4 is a RNA dependent RNA polymerase. It replicates genomic and antigenomic RNA by recognizing replications specific signals. Transcribes also a 26S subgenomic mRNA by initiating RNA synthesis internally on antigenomic RNA. This 26S mRNA encodes for structural proteins. nsP4 is a short-lived protein regulated by several ways: the opal codon readthrough and degradation by ubiquitin pathway By similarity.
Catalytic activity	S-adenosyl-L-methionine + GTP = m7GTP. m7GTP + (5')pp-Pur-mRNA = diphosphate + m7G(5')ppp-Pur-mRNA. (5')ppp-mRNA + H2O = (5')pp-mRNA + phosphate. A 5'-phosphopolynucleotide + H2O = a polynucleotide + phosphate. NTP + H2O = NDP + phosphate. ATP + H2O = ADP + phosphate. Nucleoside triphosphate + RNA(n) = diphosphate + RNA(n+1).
Subunit structure	P123 interacts with nsP4; nsP1, nsP2, nsP3 and nsP4 interact with each other, and with uncharacterized host factors By similarity.
Subcellular location	Non-structural polyprotein: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Note: Located on the cytoplasmic surface of modified endosomes and lysosomes, also called cytopathic vacuoles type I (CPVI). These vacuoles contain numerous small circular invaginations (spherules) which may be the sites of RNA synthesis. P123: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. P123': Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. mRNA-capping enzyme nsP1: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host cell membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host cell projection › host filopodium By similarity. Note: In the late phase of infection, the polyprotein is quickly cleaved before localization to cellular membranes. Then a fraction of nsP1 localizes to the inner surface of the plasma membrane and its filopodial extensions By similarity. Protease nsP2: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host nucleus By similarity. Note: In the late phase of infection, the polyprotein is quickly cleaved before localization to cellular membranes. Then approximately half of nsP2 is found in the nucleus By similarity. Non-structural protein 3: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host cytoplasm By similarity. Note: In the late phase of infection, the polyprotein is quickly cleaved before localization to cellular membranes. Then nsP3 and nsP3' seems to aggregate in cytoplasm By similarity. Non-structural protein 3': Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host cytoplasm By similarity. Note: In the late phase of infection, the polyprotein is quickly cleaved before localization to cellular membranes. Then nsP3 and nsP3' seems to aggregate in cytoplasm By similarity. RNA-directed RNA polymerase nsP4: Host endosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity. Host lysosome membrane; Peripheral membrane protein; Cytoplasmic side By similarity.
Induction	Viral replication produces dsRNA in the late phase of infection, resulting in a strong activation of host EIF2AK2/PKR, leading to almost complete phosphorylation of EIF2A. This inactivates completely cellular translation initiation, resulting in a dramatic shutoff of proteins synthesis. Translation of viral non-structural polyprotein and all cellular proteins are stopped in infected cell between 2 and 4 hours post infection. Only the 26S mRNA is still translated into viral structural proteins, presumably through a unique mechanism of enhancer element which counteract the translation inhibition mediated by EIF2A. By doing this, the virus uses the cellular defense for its own advantage: shutoff of cellular translation allows to produce big amounts of structural proteins needed for the virus to bud out of the doomed cell.
Post-translational modification	Specific enzymatic cleavages in vivo yield mature proteins. The polyprotein is synthesized as P123, or P1234 by stop codon readthrough. These polyproteins are processed differently depending on the stage of infection. In early stages, P1234 is first cleaved in trans, through its nsP2 protease activity, releasing P123' and nsP4. P123/P123' and nsP4 start to replicate the viral genome into its antigenome. After these early events, nsP1 is cleaved in cis by nsP2 protease, releasing the P23/P23' polyprotein. Cleavage of nsP1 exposes an 'activator' at the N-terminus of P23/P23' which induces its cleavage into nsP2 and nsP3 by the viral protease. This sequence of delayed processing would allow correct assembly and membrane association of the RNA-polymerase complex. In the late stage of infection, the presence of free nsP2 in the cytoplasm cleaves P1234 quickly into P12 and P34, then into the four nsP By similarity. nsP1 is palmitoylated by host By similarity. nsP4 is ubiquitinated; targets the protein for rapid degradation via the ubiquitin system By similarity.
Miscellaneous	The genome encodes for P123, but readthrough of a terminator codon UGA occurs between the codons for Asn-1852 and Arg-1853. This readthrough produces P1234, cleaved quickly by nsP2 into P123' and nsP4. Further processing of p123' gives nsP1, nsP2 and nsP3' which is 6 amino-acids longer than nsP3 since the cleavage site is after the readthrough. This unusual molecular mechanism ensures that few nsP4 are produced compared to other non-structural proteins. Mutant viruses with no alternative termination site grow significantly slower than wild-type virus.
Sequence similarities	Contains 1 Macro domain. Contains 1 peptidase C9 domain. Contains 1 RdRp catalytic domain.

Ontologies

Keywords
Biological process	RNA replication mRNA capping mRNA processing
Cellular component	Host cell membrane Host cell projection Host cytoplasm Host endosome Host lysosome Host membrane Host nucleus Membrane
Coding sequence diversity	RNA suppression of termination
Ligand	ATP-binding GTP-binding Nucleotide-binding RNA-binding
Molecular function	Helicase Hydrolase Methyltransferase Nucleotidyltransferase Protease RNA-directed RNA polymerase Thiol protease Transferase
PTM	Lipoprotein Palmitate Ubl conjugation
Technical term	Complete proteome Multifunctional enzyme
Gene Ontology (GO)
Biological process	mRNA capping Inferred from electronic annotation. Source: UniProtKB-KW proteolysis Inferred from electronic annotation. Source: UniProtKB-KW transcription, DNA-dependent Inferred from electronic annotation. Source: InterPro viral genome replication Inferred from electronic annotation. Source: InterPro
Cellular component	host cell endosome membrane Inferred from electronic annotation. Source: UniProtKB-SubCell host cell filopodium Inferred from electronic annotation. Source: UniProtKB-SubCell host cell lysosomal membrane Inferred from electronic annotation. Source: UniProtKB-SubCell host cell nucleus Inferred from electronic annotation. Source: UniProtKB-SubCell host cell plasma membrane Inferred from electronic annotation. Source: UniProtKB-SubCell membrane Inferred from electronic annotation. Source: UniProtKB-KW
Molecular function	ATP binding Inferred from electronic annotation. Source: UniProtKB-KW GTP binding Inferred from electronic annotation. Source: UniProtKB-KW RNA binding Inferred from electronic annotation. Source: UniProtKB-KW RNA-directed RNA polymerase activity Inferred from electronic annotation. Source: UniProtKB-KW cysteine-type peptidase activity Inferred from electronic annotation. Source: UniProtKB-KW helicase activity Inferred from electronic annotation. Source: UniProtKB-KW methyltransferase activity Inferred from electronic annotation. Source: UniProtKB-KW polynucleotide 5'-phosphatase activity Inferred from electronic annotation. Source: EC
Complete GO annotation...

Sequence annotation (Features)

	Feature key	Position(s)	Length	Description	Graphical view	Feature identifier
Molecule processing
	Chain	1 – 2466	2466	Non-structural polyprotein		PRO_0000308408
	Chain	1 – 1858	1858	P123'		PRO_0000228801
	Chain	1 – 1852	1852	P123		PRO_0000228802
	Chain	1 – 533	533	mRNA-capping enzyme nsP1		PRO_0000228803
	Chain	534 – 1327	794	Protease nsP2		PRO_0000228804
	Chain	1328 – 1852	525	Non-structural protein 3		PRO_0000228805
	Chain	1348 – 1858	511	Non-structural protein 3'		PRO_0000228806
	Chain	1859 – 2466	608	RNA-directed RNA polymerase nsP4		PRO_0000228807
Regions
	Domain	1001 – 1320	320	Peptidase C9
	Domain	1328 – 1486	159	Macro
	Domain	2223 – 2338	116	RdRp catalytic
	Nucleotide binding	719 – 726	8	ATP Potential
	Region	242 – 261	20	nsP1 membrane-binding By similarity
	Region	1002 – 1021	20	Nucleolus localization signal By similarity
	Motif	1177 – 1181	5	Nuclear localization signal By similarity
Sites
	Active site	1010	1	For cysteine protease nsP2 activity By similarity
	Active site	1079	1	For cysteine protease nsP2 activity By similarity
	Site	533 – 534	2	Cleavage; by nsP2 By similarity
	Site	1327 – 1328	2	Cleavage; by nsP2 By similarity
	Site	1858 – 1859	2	Cleavage; by nsP2 By similarity
Amino acid modifications
	Lipidation	417	1	S-palmitoyl cysteine; by host By similarity
Natural variations
	Natural variant	2460	1	S → N in strain: BFS1703.

Sequences

	Sequence		Length	Mass (Da)
	P13896 [UniParc]. Last modified March 21, 2006. Version 2. Checksum: 646CB1A22FB63289	FASTA	2,466	274,991
	10 20 30 40 50 60 MERIHVDLDA DSPYVKSLQR TFPQFEIEAR QVTDNDHANA RAFSHVATKL IESEVDRDQV 70 80 90 100 110 120 ILDIGSAPVR HAHSNHRYHC ICPMISAEDP DRLQRYAERL KKSDITDKNI ASKAADLLEV 130 140 150 160 170 180 MSTPDAETPS LCMHTDATCR YFGSVAVYQD VYAVHAPTSI YHQALKGVRT IYWIGFDTTP 190 200 210 220 230 240 FMYKNMAGSY PTYNTNWADE RVLEARNIGL GNSDLQESRL GKLSILRKKR LQPTNKIIFS 250 260 270 280 290 300 VGSTIYTEDR SLLRSWHLPN VFHLKGKSNF TGRCGTIVSC EGYVIKKITI SPGLYGKVEN 310 320 330 340 350 360 LASTMHREGF LSCKVTDTLR GERVSFAVCT YVPATLCDQM TGILATDVSV DDAQKLLVGL 370 380 390 400 410 420 NQRIVVNGRT QRNTNTMQNY LLPVVAQAFS RWAREHRADL DDEKELGVRE RTLTMGCCWA 430 440 450 460 470 480 FKTQKITSIY KKPGTQTIKK VPAVFDSFVI PRLTSHGLDM GFRRRLKLLL EPTVKPAPAI 490 500 510 520 530 540 TMADVEHLRG LQQEAEEVAA AEEIREALPP LLPEIEKETV EAEVDLIMQE AGAGSVETPR 550 560 570 580 590 600 GHIRVTSYPG EEKIGSYAIL SPQAVLNSEK LACIHPLAEQ VLVMTHKGRA GRYKVEPYHG 610 620 630 640 650 660 KVIVPEGTAV PVQDFQALSE SATIVFNERE FVNRYLHHIA INGGALNTDE EYYKTVKTQD 670 680 690 700 710 720 TDSEYVFDID ARKCVKREDA GPLCLTGDLV DPPFHEFAYE SLKTRPAAPH KVPTIGVYGV 730 740 750 760 770 780 PGSGKSGIIK SAVTKKDLVV SAKKENCAEI IRDVRRMRRM DVAARTVDSV LLNGVKHPVN 790 800 810 820 830 840 TLYIDEAFAC HAGTLLALIA IVKPKKVVLC GDPKQCGFFN MMCLKVHFNH DICTEVYHKS 850 860 870 880 890 900 ISRRCTQTVT AIVSTLFYDK RMKTVNPCAD KIIIDTTGTT KPHKDDLILT CFRGWVKQLQ 910 920 930 940 950 960 IDYKNHEIMT AAASQGLTRK GVYAVRYKVN ENPLYSQTSE HVNVLLTRTE KRIVWKTLAG 970 980 990 1000 1010 1020 DPWIKTLTAK YPGDFTASLD DWQREHDAIM ARVLDKPQTA DVFQNKVNVC WAKALEPVLA 1030 1040 1050 1060 1070 1080 TANIVLTRQQ WETLHPFKHD RAYSPEMALN FFCTRFFGVD LDSGLFSAPT VALTYRDQHW 1090 1100 1110 1120 1130 1140 DNSPGKNMYG LNREVAKELS RRYPCITKAV DTGRVADIRN NTIKDYSPTI NVVPLNRRLP 1150 1160 1170 1180 1190 1200 HSLIVDHKGQ GTTDHSGFLS KMKGKSVLVI GDPISIPGKK VESMGPLPTN TIRCDLDLGI 1210 1220 1230 1240 1250 1260 PSHVGKYDII FVNVRTPYRN HHYQQCEDHA IHHSMLTCKA VHHLNTGGTC VAIGYGLADR 1270 1280 1290 1300 1310 1320 ATENIITAVA RSFRFTRVCQ PKNTAENTEV LFVFFGKDNG NHTHDQDRLG VVLDNIYQGS 1330 1340 1350 1360 1370 1380 TRYEAGRAPA YRVIRGDISK SADQAIVNAA NSKGQPGSGV CGALYRKWPA AFDRQPIAVG 1390 1400 1410 1420 1430 1440 TARLVKHEPL IIHAVGPNFS KMPEPEGDLK LAAAYMSIAS IVNAERITKI SVPLLSTGIY 1450 1460 1470 1480 1490 1500 SGGKDRVMQS LHHLFTAFDT TDADVTIYCL DKQWETRIIE AIHRKESVEI LDDDKPVDID 1510 1520 1530 1540 1550 1560 LVRVHPNSSL AGRPGYSVNE GKLYSYLEGT RFHQTAKDIA EIHAMWPNKS EANEQICLYI 1570 1580 1590 1600 1610 1620 LGESMSSIRS KCPVEESEAS APPHTLPCLC NYAMTAERVY RLRSAKKEQF AVCSSFLLPK 1630 1640 1650 1660 1670 1680 YRITGVQKLQ CSKPVLFSGV VPPAVHPRKY AEIILETPPP PATTTVICEP TVPERIPSPV 1690 1700 1710 1720 1730 1740 ISRAPSAESL LSLGGVSFSS SATRSSTAWS DYDRRFVVTA DVHQANTSTW SIPSAPGLDV 1750 1760 1770 1780 1790 1800 QLPSDVTDSH WSIPSASGFE VRTPSVQDLT AECAKPRGLA EIMQDFNTAP FQFLSDYRPV 1810 1820 1830 1840 1850 1860 PAPRRRPIPS PRSTASAPPV PKPRRTKYQQ PPGVARAISE AELDEYIRQH SNRYEAGAYI 1870 1880 1890 1900 1910 1920 FSSETGQGHL QQKSVRQCKL QEPILDRAVH EKYYAPRLDL EREKMLQKKL QLCASEGNRS 1930 1940 1950 1960 1970 1980 RYQSRKVENM KAITAERLIS GLGTYLSSEV NPVECYRVNY PVPIYSSTVI NRFTSAEVAV 1990 2000 2010 2020 2030 2040 KTCNLVIQEN YPTVASYCIT DEYDAYLDMV DGASCCLDTA TFCPAKLRSY PKKHSYLQPE 2050 2060 2070 2080 2090 2100 IRSAVPSPIQ NTLQNVLAAA TKRNCNVTQM RELPVLDSAA FNVDCFKKYA CNDEYWDTFR 2110 2120 2130 2140 2150 2160 DNPIRLTTEN VTQYVTKLKG PKAAALFANT HNLKPLQEIP MDQFVMDLKR DVKVTPGTKH 2170 2180 2190 2200 2210 2220 TEERPKVQVI QAADPLATAY LCGIHRELVR RLNAVLLPNI HTLFDMSAED FDAIIAEHFH 2230 2240 2250 2260 2270 2280 HGDPVLETDI ASFDKSEDDA IAISALMILE DLGVDQPLLD LIEAAFGNIT SVHLPTGTRF 2290 2300 2310 2320 2330 2340 KFGAMMKSGM FLTLFVNTLV NIMIASRVLR ERLTTSACAA SIGDDNIVHG VVSDTLMAER 2350 2360 2370 2380 2390 2400 CATWLNMEVK IIDAVIGIKA PYFCGGFILV DQITGTACRV ADPLKRLFKL GKPLPVDDTQ 2410 2420 2430 2440 2450 2460 DCDRRRALHD EAMRWNRIGI TDELVKAVES RYEIILAGLI ITSLSTLAES VKNFKSIRGS PITLYG

References

[1]	"Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes." Netolitzky D.J., Schmaltz F.L., Parker M.D., Rayner G.A., Fisher G.R., Trent D.W., Bader D.E., Nagata L.P. J. Gen. Virol. 81:151-159(2000) [PubMed: 10640553] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC RNA].
[2]	"Western equine encephalitis virus is a recombinant virus." Hahn C.S., Lustig S., Strauss E.G., Strauss J.H. Proc. Natl. Acad. Sci. U.S.A. 85:5997-6001(1988) [PubMed: 3413072] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC RNA] OF 2430-2466. Strain: BFS1703.

Cross-references

Sequence databases
EMBL GenBank DDBJ	AF214040 Genomic RNA. Translation: AAF28339.1. J03854 Genomic RNA. Translation: AAA42998.1.
RefSeq	NP_640330.1. NC_003908.1.
3D structure databases
ProteinModelPortal	P13896.
SMR	P13896. Positions 1001-1320.
ModBase	Search...
Protein family/group databases
MEROPS	C09.002.
Protocols and materials databases
StructuralBiologyKnowledgebase	Search...
Genome annotation databases
GeneID	944530.
Family and domain databases
InterPro	IPR002589. A1pp. IPR000606. Helicase_domain_viral-like. IPR002620. Pept_C9_alphavirus_nsp2pro. IPR007094. RNA-dir_pol_PSvirus. IPR001788. Tymovirus_RNA-dep_RNA_pol. [Graphical view]
Pfam	PF01661. Macro. 1 hit. PF01707. Peptidase_C9. 1 hit. PF00978. RdRP_2. 1 hit. PF01443. Viral_helicase1. 1 hit. [Graphical view]
SMART	SM00506. A1pp. 1 hit. [Graphical view]
PROSITE	PS51154. MACRO. 1 hit. PS51520. NSP2PRO. 1 hit. PS50507. RDRP_SSRNA_POS. 1 hit. [Graphical view]
ProtoNet	Search...

Entry information

Entry name

POLN_WEEV

Accession

Primary (citable) accession number: P13896
Secondary accession number(s): Q9J1K2

Entry history

Integrated into UniProtKB/Swiss-Prot:	January 1, 1990
Last sequence update:	March 21, 2006
Last modified:	November 16, 2011
This is version 79 of the entry and version 2 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Viral Protein Annotation Program