Viral RNA and Proteome

Viral RNA

Viral Proteome

• NSP 1 is the major pathogenic/ (cell death inducing ) gene of the virus. Works by keeping Ribosome 40S RNA entry latch closed and makes it impossible for the host mRNA to be loaded . Thereby, inhibiting host RNA translation but allowing viral RNA translation through the use of viral 5′ UTR sequences . Induces an endonucleolytic cleavage near the 5’UTR of host mRNAs, targeting them for degradation. Viral mRNAs are not susceptible to nsp1-mediated endonucleolytic RNA cleavage thanks to the presence of a 5′-end leader sequence and are therefore protected from degradation. Binding studies suggest interactions with DNA polymerase alpha/primase complex (PRIM1, PRIM2, POLA1, POLA2) , COLGALT1 and PKP2. Also bind 5_SL1.

• NSP 2 – binds PHB, PHB2, EIF4E2, FKBP15, GIGYF2, POR, WASHC4, RAP1GDS1, and SLC27A2
• NSP 3/Papain Like Proteinase -Responsible for the cleavages located at the N-terminus of the replicase polyprotein. Participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Antagonizes innate immune induction of type I interferon by blocking the phosphorylation, dimerization and subsequent nuclear translocation of host IRF3. Prevents host NF-kappa-B signaling . In addition, possesses a deubiquitinating/deISGylating activity and processes both ‘Lys-48’- and ‘Lys-63’-linked polyubiquitin chains from cellular substrates. Preferentially cleaves ISG15 from substrates in vitro and avoids MDA-5 + ISG15 oligomerization and stimulation of MAV based antiviral defenses.. Can play a role in host ADP-ribosylation by binding ADP-ribose. Forms the pore of the DMV for RTC transport of + viral RNA into the cytoplasm where it is encapsulated by the Nucleocapsid protein.
• NSP 4 – Participates in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. binds TIM complex proteins (TIMM10, TIMM10b, TIMM29, TIMM9), NUP210, IDE, ALG11, and DNAJC11 . Has 4 transmembrane spanning segments. Part of the RTC Pore that provides transit of + RNA from Double membrane vesicle lumens into the cytoplasm.
• 3 CL Proteinase / NSP 5 – Cleaves the C-terminus of replicase polyprotein at 11 sites . Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN]. Also able to bind an ADP-ribose-1”-phosphate (ADRP).  binds HDAC2 or version C145A binds TRMT1 and GPX1
• NSP 6 -Plays a role in the initial induction of autophagosomes from host endoplasmic reticulum. Later, limits the expansion of these phagosomes that are no longer able to deliver viral components to lysosomes. binds SIGMAR1, ATP13A3, ATP5MG, ATP6AP1 . Has 6 transmembrane spanning segments.
• NSP 7 – Forms a hexadecamer with NSP 8. Shows primase activity. binds Rab signaling proteins ( RAB18, RAB14, RAB8A, RAB7A, RAB10, RAB5C, RAB2A, RAB1A) and ( LMAN2, GNG5, GNB1, NDUFAF2, COMT, HS2ST1, QSOX2, RALA, RHOA, MTARC1, SCCPDH, SELENOS, AGPS, TOR1AIP1, DNAJC19, MOGS, ACSL3, FAM162A, DCAKD, NAT14, SCARB1, CYB5B, PTGES2, CYB5R3, )
• NSP 8 – Forms a hexadecamer with NSP 7. Shows primase activity . binds Exosome Complex proteins (EXOSC2, EXOSC3, EXOSC5, EXOSC8), 7SK snRP ( LARP7, MEPCE), Signal Recognition Particle (SRP19, SRP72, SRP54), Mitochondrial Ribosome (MRPS2, MRPS5, MRPS25, MRPS27), and other proteins (SEPSECS, ATE1, NSD2, MPHOSPH10 , DDX10, HECTD1, NGDN, NOL10, AATF, NARS2, CCDC86) Binds 3_SL3 flanking sequences associated with replication initiation.
• NSP 9 – May participate in viral replication by acting as a ssRNA-binding protein . Also binds Nuclear pore proteins ( NUP58, NUP86, NUP14, NUP62, NUP214), Fibrillin (FBLN5, FBN1, FBN2), and (EIF4H, DCAF7, GTF2F2, NEK9, MIB1, SPART, ZNF503)
• NSP 10 -Plays a pivotal role in viral transcription by stimulating both nsp14 3′-5′ exoribonuclease and nsp16 2′-O-methyltransferase activities. Therefore plays an essential role in viral mRNAs cap methylation. Also binds to AP2 Clathrin (AP2A2, AP2M1 ) , GRPEL1, GFER, ERGIC1
• NSP 11 – binds TBCA

• RNA Depenedent RNA Polymerase / NSP 12 -binds RIPK1, RBM41, PRRC2B, PLEKHA5, PDZD11, MYCBP2, LARP4B, CRTC3 , AKAP8, UBAP2L, ZNF318, ZC3H7A, BCKDK, USP54, TYSND1, TCF12, SBNO1, PPIL3, SLU7 .
• Helicase /NSP 13 – binds Golgi apparatus proteins ( GOLGA2, GOLGA3, GOLGB, GORASP1, GCC2, GCC1) , TLE family (TLE1, TLE3, TLE5) , Centrosome ( NIN, NINL, PCNT, CDK5RAP2, CENPF, CEP112, CEP135, CEP250, CDP350, CEP43, CEP68,CNTRL ) , protein kinase A signaling (PRKACA, AKAP9, PRKAR2A, PRKAR2B) and others (ERG4, C1ORF50, RDX, CLIP4, JAKMIP1, GRIPAP1, TBK1, TBKBP1, CIT, MIPOL1, USP13, HSBP1, FYCO1, PDE4DIP, HOOK1)
• NSP 14 3′-5′ exoribonuclease  Binds GLA, SIRT5, IMPDH2
• NSP 15 – Uridylate-specific endoribonuclease. Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2′-3′-cyclic phosphates 5′ to the cleaved bond . Binds NUTF2, RNF41, ARF6
• NSP 16 -m2′-O-methyltransferase involved in mRNA cap methylation.

• Spike protein – binds protein palmitoylation proteins ( GOLGA7, ZDHHC5)
• Orf 3a – Forms homotetrameric potassium sensitive ion channels (viroporin) and may modulate virus release. Up-regulates expression of fibrinogen subunits FGA, FGB and FGG in host lung epithelial cells. Induces apoptosis in cell culture. Downregulates the type 1 interferon receptor by inducing serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and increasing IFNAR1 ubiquitination Involved in escort independent budding. binds HOPS complex, and STOML2. Localizes to lysosomes and promotes deacidification and decreased activity of lysosomal proteases.
• Orf 3d (Overlapping gene lacking T4 and T8 epitopes)
• Orf 3c ( Overlapping gene)
• Envelope protein Central role in viral morphogenesis and assembly. Interacts with Membrane protein. Behaves like a viroporin. Promotes apoptosis. . Binds to BRD2, BRD4, CWC27, AP3B1, ZC3H18 , SLC44A2.
• Membrane protein – Initiates viral morphogenesis and assembly via contact with viral RNP nucleocapsid protein. Initial viral particle invagination occurs in the ER or ERGIC. Also Interacts with envelope proteins. Binds solute carrier family proteins (SLC25A21,SLC30A7, SLC30A9), ER morphology proteins (REEP6, REEP5, YIF1A, RTN4) and (SAAL1, AAR2, TARS2, STOM, PSMD8, PMPCB, PMPCA, PITRM1, INTS4, GGCX, FASTKD5, FAM8A1, ETFA, COQ8B, BZW2, ATP6V1A, ATP1B1, AASS, ACADM, AKAP8L, ANO6, TUBGCP3, TUBGCP2)
• Orf 6 – Disrupts interferon signaling by preventing nuclear import of STAT proteins via binding to Nup98.-Rae nuclear pore complex. Also binds to and tethers karyopherin alpha 2 and karyopherin beta 1 to the ER/Golgi membranes. Also binds to the proapoptotic MTCH1 mitochondrial OMM protein.
• Orf 7a Disrupts tetherin antiviral activity Binds HEATR3, MDN1
• Orf 7b Interacts with MAVS and UNC93B1
• Orf 8 – Inhibition of MHC Class I presentation. Binds ER protein quality control proteins (EDEM3, ERLEC1, OS9, UGGT2, ERO1B, SIL1, HYOU1, NGLY1, TOR1A, FOXRED2, SDF2 ), effects Cholesterol metabolism ( NPC2, PCSK6 ), and (ADAMTS1, TM2D3, CHPF, LOX, PVR, COL6A1, PLOD2, FKBP10, FKBP7, GGH, IL17RA, PUSL1, PLD3, ADAM9, ITGB1, DNMT1, EMC1, CHPF2, ERP44, MFGE8, HS6ST2, POFUT1, NPTX1, STC2, POGLUT2, PLAT, NEU1, SMOC1, GDF15, INHBE, CISD3, PLEKHF2, POGLUT3, FBXL12 .
• Orf 9b (Alternative Orf in N gene) – Disrupts RiG like receptor based innate immunity. binds MARK kinases (MARK1, MARK2, MARK3), DCTPP1, TOMM70, SLC9A3R1, CHMP2A, DPH5, CSDE1, PTBP2, BAG5.
• Orf 9c – binds Electron transport proteins (ECSIT, NDUFAF1, NDUFB9, ACAD9) and GPI anchor biosynthesis enzymes ( GPAA1, PIGS). BCS1L, SCAP, TMED5, SLC30A6, NDFIP2, DPY19L1, F2RL1, GHITM, ABCC1, TMEM39B, ALG8, FAR2, WFS1, PIGO, RETREG3, UBXN8, NLRX1, TMEM97, ERMP1, TAPT1.
• Nucleocapsid – binds stress granules (G3BP1, G3BP2, CSNK2A2, CSNK2B, SNIP1, FAM98B )and RNA processing proteins ( LARP1, PABPC1, MOV10, RPL36, RBM28, DDX21, UPF1, RRP9, PABPC4). Binds 5_SL6 and 3′ SL’s , Phosphorylated by GSK 3 and cdk1 kinases which enhances the formation of liquid droplets and release of tightly bound viral RNA. Interacts with the UBL1 domain of NSP3 and may mediate encapsidation of + RNA emanating from DMVs.
• Orf 10 – ? if expressed . binds CUL2 complex (CUL2, ZYG11B, RBX1, ELOB, ELOC), and (TIMM8B, THTPA, MAP7D1, PPT1)
• Orf 14

COVID 19 NMR Project