Reviewed, UniProtKB/Swiss-Prot P36327 (POLN_EEVV3)

Names and origin

Protein names	Recommended name:     Non-structural polyprotein Alternative name(s):     Polyprotein nsP1234       Short name=P1234 Cleaved into the following 7 chains:     1- Recommended name:             P123     2- Recommended name:             P123'     3- Recommended name:             mRNA-capping enzyme nsP1               EC=2.1.1.-               EC=2.7.7.-         Alternative name(s):             Non-structural protein 1     4- Recommended name:             Protease/triphosphatase/NTPase/helicase nsP2               EC=3.4.22.-               EC=3.1.3.33               EC=3.6.1.15               EC=3.6.1.-         Alternative name(s):             Non-structural protein 2               Short name=nsP2     5- Recommended name:             Non-structural protein 3                 Short name=nsP3     6- Recommended name:             Non-structural protein 3'                 Short name=nsP3'     7- Recommended name:             RNA-directed RNA polymerase nsP4               EC=2.7.7.48         Alternative name(s):             Non-structural protein 4               Short name=nsP4
Organism	Venezuelan equine encephalitis virus (strain 3880) (VEEV)
Taxonomic identifier	36382 [NCBI]
Taxonomic lineage	Viruses › ssRNA positive-strand viruses, no DNA stage › Togaviridae › Alphavirus › VEEV complex
Virus host	Didelphis marsupialis (Southern opossum) [TaxID: 9268] Philander opossum (Gray four-eyed opossum) [TaxID: 9272] Homo sapiens (Human) [TaxID: 9606] Equus asinus (Donkey) [TaxID: 9793] Equus caballus (Horse) [TaxID: 9796] Bos taurus (Bovine) [TaxID: 9913] Proechimys [TaxID: 10162] Sigmodon hispidus (Hispid cotton rat) [TaxID: 42415] Melanoconion [TaxID: 53535]

Protein attributes

Sequence length	2485 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. 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.
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/triphosphatase/NTPase/helicase 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 Gln-1871 and Arg-1872. This readthrough produces P1234, cleaved quickly by nsP2 into P123' and nsP4. Further processing of p123' gives nsP1, nsP2 and nsP3' which is 7 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 By similarity.
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	Cell membrane Cell projection Cytoplasm Endosome Lysosome Membrane Nucleus
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 Phosphoprotein Ubl conjugation
Technical term	Multifunctional enzyme
Gene Ontology (GO)
Biological process	mRNA capping Inferred from electronic annotation. Source: UniProtKB-KW transcription, RNA-dependent Inferred from electronic annotation. Source: UniProtKB-KW viral genome replication Inferred from electronic annotation. Source: InterPro
Cellular component	cell projection Inferred from electronic annotation. Source: UniProtKB-KW endosome Inferred from electronic annotation. Source: UniProtKB-KW host cell cytoplasm 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 lysosome Inferred from electronic annotation. Source: UniProtKB-KW nucleus Inferred from electronic annotation. Source: UniProtKB-KW plasma 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 helicase activity Inferred from electronic annotation. Source: InterPro RNA-directed RNA polymerase activity Inferred from electronic annotation. Source: UniProtKB-KW cysteine-type endopeptidase activity Inferred from electronic annotation. Source: InterPro 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 – 2485	2485	Non-structural polyprotein		PRO_0000308387
	Chain	1 – 1878	1878	P123'		PRO_0000228750
	Chain	1 – 1871	1871	P123		PRO_0000228751
	Chain	1 – 535	535	mRNA-capping enzyme nsP1		PRO_0000041200
	Chain	536 – 1329	794	Protease/triphosphatase/NTPase/helicase nsP2		PRO_0000041201
	Chain	1330 – 1878	549	Non-structural protein 3'		PRO_0000228752
	Chain	1330 – 1871	542	Non-structural protein 3		PRO_0000041202
	Chain	1879 – 2485	607	RNA-directed RNA polymerase nsP4		PRO_0000041203
Regions
	Domain	963 – 1163	201	Peptidase C9
	Domain	1330 – 1489	160	Macro
	Domain	2242 – 2357	116	RdRp catalytic
	Nucleotide binding	721 – 728	8	ATP Potential
	Region	244 – 263	20	nsP1 membrane-binding By similarity
	Region	1004 – 1023	20	Nucleolus localization signal By similarity
	Motif	1179 – 1183	5	Nuclear localization signal By similarity
Sites
	Active site	1012	1	For cysteine protease nsP2 activity By similarity
	Active site	1081	1	For cysteine protease nsP2 activity By similarity
	Site	535 – 536	2	Cleavage; by nsP2 By similarity
	Site	1329 – 1330	2	Cleavage; by nsP2 By similarity
	Site	1878 – 1879	2	Cleavage; by nsP2 By similarity
Amino acid modifications
	Lipidation	419	1	S-palmitoyl cysteine; by host By similarity

Sequences

	Sequence		Length	Mass (Da)
	P36327-1 [UniParc]. Last modified May 30, 2000. Version 2. Checksum: 83458A964660D639	FASTA	2,485	277,285
	10 20 30 40 50 60 MEKVHVDIEE DSPFLRALQR SFPQFEVEAK QVTDNDHANA RAFSHLASKL IETEVDPSDT 70 80 90 100 110 120 ILDIGSAPAR RMYSKHKYHC ICPMRCAEDP DRLYKYATKL KKNCKEITDK ELDKKMKELA 130 140 150 160 170 180 AVMSDPDLET ETMCLHDDES CRYEGQVAVY QDVYAVDGPT SLYHQANKGV RVAYWIGFDT 190 200 210 220 230 240 TPFMFKNLAG AYPSYSTNWA DETVLTARNI GLCSSDVMER SRRGMSILRK KYLKPSNNVL 250 260 270 280 290 300 FSVGSTIYHE KRDLLRSWHL PSVFHLRGKQ NYTCRCETIV SCDGYVVKRI AISPGLYGKP 310 320 330 340 350 360 SGYAATMHRE GFLCCKVTDT LNGERVSFPV CTYVPATLCD QMTGILATDV SADDAQKLLV 370 380 390 400 410 420 GLNQRIVVNG RTQRNTNTMK NYLLPVVAQA FARWAKEYKE DQEDERPLGL RDRQLVMGCC 430 440 450 460 470 480 WAFRRHKITS IYKRPDTQTI IKVNSDFHSF VLPRIGSNTL EIGLRTRIRK MLEEHKEPSP 490 500 510 520 530 540 LITAEDIQEA KCAADEAKEV REAEELRAVL PPLAADVEEP TLEADVDLML QEAGAGSVET 550 560 570 580 590 600 PRGLIKVTSY AGEDKIGSYA VLSPQAVLKS EKLSCIHPLA EQVIVITHSG RKGRYAVEPY 610 620 630 640 650 660 HGKVVVPEGH AIPVQDFQAL SESATIVYNE REFVNRYLHH IATHGGALNT DEEYYKTVKP 670 680 690 700 710 720 SEHDGEYLYD IDRKQCVKKE LVTGLGLTGE LVDPPFHEFA YESLRTRPAA PYQVPTIGVY 730 740 750 760 770 780 GVPGSGKSGI IKSAVTKKDL VVSAKKENCA EIIRDVKRIK GLDVNARTVD SVLLNGCKYP 790 800 810 820 830 840 VETLYIDEAF ACHAGTLRAL IAIIRPKKAV LCGDPKQCGF FNMMCLKVHF NHEICTQVFH 850 860 870 880 890 900 KSISRRCTKS VTSVVSTLFY DKRMRTTNPK ETKIEIDTTG STKPKQDDLI LTCFRGWVKQ 910 920 930 940 950 960 LQIDYKGNEV MTAAASQGLT RKGVYAVRYK VNENPLYAPT SEHVNVLLTR TEDRIVWKTL 970 980 990 1000 1010 1020 AGDPWIKTLT AKYPGNFTAT IEEWQAEHDA IMRHILERPD PTDVFQNKAN VCWAKALVPV 1030 1040 1050 1060 1070 1080 LKTAGIDMTT EQWNTVDYFE TDKAHSAEIV LNQLCVRFFG LDLDSGLFSA PTVPLSIRNN 1090 1100 1110 1120 1130 1140 HWDNSPSPNM YGLNKEVVRQ LSRRYPQLPR AVTTGRVYDM NTGTLRNYDP RINLVPVNRR 1150 1160 1170 1180 1190 1200 LPHALVLHHN EHPQSDFSSF VSKLKGRTVL VVGEKLSVPG KTVDWLSDRP EATFRARLDL 1210 1220 1230 1240 1250 1260 GIPGDVPKYD IIFINVRTPY KYHHYQQCED HAIKLSMLTK KACLHLNPGG TCVSIGYGYA 1270 1280 1290 1300 1310 1320 DRASESIIGA IARQFKFSRV CKPKSSLEET EVLFVFIGYD RKARTHNPYK LSSTLTNIYT 1330 1340 1350 1360 1370 1380 GSRLHEAGCA PSYHVVRGDI ATATEGVIIN AANSKGQPGG GVCGALYKKF PESFDLQPIE 1390 1400 1410 1420 1430 1440 VGKARLVKGA AKHIIHAVGP NFNKVSEIEG DKQLAEAYES IAKIVNDNNY KSVAIPLLST 1450 1460 1470 1480 1490 1500 GIFSGNKDRL TQSLNHLLTA LDTTDADVAI YCRDKKWEMT LKEAVARREA VEEICISDDS 1510 1520 1530 1540 1550 1560 SVTEPDAELV RVHPKSSLAG RKGYSTSDGK TFSYLEGTKF HQAAKDIAEI NAMWPVATEA 1570 1580 1590 1600 1610 1620 NEQVCMYILG ESMSSIRSKC PVEESEASTP PSTLPCLCIH AMTPERVQRL KASRPEQITV 1630 1640 1650 1660 1670 1680 CSSFPLPKYR ITGVQKIQCS QPILFSPKVP AYIHPRKYLV ETPTVEENQS TEGTPEQPTL 1690 1700 1710 1720 1730 1740 ITVGETRTRT PEPIIIEEEE DSISLLSDGP THQVLQVEAD IHGPPSASSS SWSIPHASDF 1750 1760 1770 1780 1790 1800 DVDSLSILDT LEGASVTSEE ASVETNSHFA RSMEFLARPV PAPRTVFRNP PQPAPRTRTP 1810 1820 1830 1840 1850 1860 SLAPSRASSR ISLVSNPPGV NRVITREELE ALTPSRAPSR SVSRTSLVSN PPGVNRVITR 1870 1880 1890 1900 1910 1920 EEFEAFVAQQ QRRFDAGAYI FSSDTGQGHL QQKSVRQTVL SEVVLERTEL EISYAPRLDQ 1930 1940 1950 1960 1970 1980 EKEELLRKKL QLNPTPANRS RYQSRRVENM KAITARRILQ GLGHYLKAEG KVECYRTLHP 1990 2000 2010 2020 2030 2040 VPLYSSSVNR AFSSPKVAVE ACNAMLKENF PTVASYCIIP EYDAYLDMVD GASCCLDTAS 2050 2060 2070 2080 2090 2100 FCPAKLRSFP KKHSYLEPTI RSAVPSAIQN TLQNVLAAAT KRNCNVTQMR ELPVLDSAAF 2110 2120 2130 2140 2150 2160 NVECFKKYAC NNEYWETFKE NPIRLTEENV VNYITKLKGP KAAALFAKTH NLNMLQDIPM 2170 2180 2190 2200 2210 2220 DRFVMDLKRD VKVTPGTKHT EERPKVQVIQ AADPLATAYL CGIHRELVRR LNAVLLPNIH 2230 2240 2250 2260 2270 2280 TLFDMSAEDF DAIIAEHFQP GDCVLETDIA SFDKSEDDAM ALTALMILED LGVDAELLTL 2290 2300 2310 2320 2330 2340 IEAAFGEISS IHLPTKTKFK FGAMMKSGMF LTLFVNTVIN IVIASRVLRE RLTGSPCAAF 2350 2360 2370 2380 2390 2400 IGDDNIVKGV KSDKLMADRC ATWLNMEVKI IDAVVGEKAP YFCGGFILCD SVTGTACRVA 2410 2420 2430 2440 2450 2460 DPLKRLFKLG KPLAADDEHD DDRRRALHEE STRWNRVGIL PELCKAVESR YETVGTSIIV 2470 2480 MAMTTLASSV KSFSYLRGAP ITLYG

References

[1]	"Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus." Kinney R.M., Tsuchiya K.R., Sneider J.M., Trent D.W. Virology 191:569-580(1992) [PubMed: 1448915] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC RNA].

Cross-references

Sequence databases
EMBLGenBankDDBJ	L00930 Genomic RNA. Translation: AAC19324.1. L00930 Genomic RNA. Translation: AAC19326.1. Sequence problems.
PIR	C44213.
3D structure databases
HSSP	HSSP built from PDB template 1FW5 based on UniProtKB P08411.
SMR	P36327. Positions 1003-1322.
ModBase	Search...
Family and domain databases
InterPro	IPR002589. A1pp. IPR002620. Peptidase_C9. IPR001788. RNA-dep_RNA_pol_vir-typ. IPR000606. RNA_helicase1_vir. IPR007094. RNA_pol_PSvir. [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. PS50507. RDRP_SSRNA_POS. 1 hit. [Graphical view]
ProtoNet	Search...

Entry information

Entry name

POLN_EEVV3

Accession

Primary (citable) accession number: P36327

Entry history

Integrated into UniProtKB/Swiss-Prot:	June 1, 1994
Last sequence update:	May 30, 2000
Last modified:	June 16, 2009
This is version 70 of the entry and version 2 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation project

Virus (Virus annotation project)