key: cord-0264779-dk57ko2c authors: Prescott, Lucas title: Alphavirus nsP2 protease structure and cleavage prediction: Possible relevance to the pathogenesis of viral arthritis date: 2022-01-23 journal: bioRxiv DOI: 10.1101/2022.01.22.477317 sha: 607b7f11db4569731c840e5fbd713ff34d9b4cd9 doc_id: 264779 cord_uid: dk57ko2c Alphaviruses are a diverse genus of arboviruses capable of infecting many vertebrates including humans. Human infection is common in equatorial and subtropical regions and is often accompanied by arthralgia or encephalitis depending on viral lineage. No antivirals or vaccines have been approved, and many alphavirus lineages have only recently been discovered and classified. Alphavirus nsP2 protease is an important virulence factor yet is commonly thought to be a simple papain-like protease which only cleaves viral polyproteins. Here, I reveal novel molecular mechanisms of these proteases via sequence and predicted structure alignment and propose novel cellular mechanisms for the pathogenesis of viral arthritis by predicting which human proteins are likely cleaved by these proteases. In addition to the known primary cysteine mechanism in all alphaviruses and a secondary serine mechanism documented in chikungunya virus (CHIKV), I discovered secondary cysteine and threonine mechanisms exist in many other alphaviruses and that these secondary mechanisms coevolve with their viral polyprotein cleavages. As for cleavage prediction, neural networks trained on 93 different putative viral polyprotein cleavages achieved a Matthews correlation coefficient of 0.965, and, when applied to the human proteome, predicted that hundreds of proteins may be vulnerable. Notable pathways likely affected by cleavages include the cytoskeleton and extracellular matrix, antiproteases, protein translation/folding/glycosylation/ubiquitination, cellular differentiation, inflammation, and vesicle trafficking, hinting that this viral protease is a more important virulence factor than previously believed. turnip yellow mosaic virus (TYMV) protease is but HEV and RUBV proteases are not structurally related 108 to alphavirus nsP2pro. TYMV protease includes an equivalent catalytic cysteine helix and activity-tuning 109 histidine flexible loop[39] yet does not contain an MTase-like domain to form a cleft as in alphaviruses. 110 In addition to this more accessible active site, TYMV protease includes two hydrophobic patches 111 required for interaction with ubiquitin for its deubiquitinating activity ( Figure 3 ) Alignment of all known alphavirus proteases ( Figure 4) indicated that, in addition to a primary 123 cysteine mechanism in all alphaviruses and a secondary serine mechanism found in at least CHIKV,[5] 124 some proteases have secondary cysteine or threonine mechanisms. These secondary mechanisms may 125 restrict or extend the possible acidic residues aligning and polarizing the catalytic histidine, [43] and 126 serine and threonine mechanisms may extend catalytic activity to higher pH. [44] By investigating how 127 substrate sequences, particularly the P1 residue, coevolve with these different mechanisms, multiple 128 functional hypotheses can be proposed: (1) cleavage after a P1 cysteine is most efficient when the 129 secondary catalytic residue is another cysteine or serine, possible for an inert secondary alanine, and 130 least efficient for a secondary threonine, (2) a secondary threonine is required for cleavage after a P1 131 serine, and (3) an inert secondary alanine is required for cleavage after a P1 arginine. proximity with each other that they may bind ordered water molecules as in other proteases [45] aim its protonated side toward the catalytic cysteine, serine, or threonine, preventing the charge relay 145 mechanism required for proteolysis ( Figure 5A ). Additionally, metal binding to another histidine in the 146 center of the loop may aid its flexing backward to allow substrate loading. In some divergent, 147 unclassified alphavirus proteases, the adjacent aspartic acid is replaced with serine, but in these cases 148 there is always another nearby potential metal-binding residue (glutamic acid, aspartic acid, or another 149 histidine)( Figure 5B ). 150 151 152 contains an alanine in this position ( Figure 4 ). No sequence features obviously correlated with this 161 substitution, and ASALV protease's predicted structure is extremely similar to those of related 162 alphavirus proteases, indicating that other alphavirus proteases may also cleave alanine-containing 163 substrates albeit possibly with suboptimal kinetics. Tryptophan is typically thought not to be directly 164 involved in the active site, yet it appeared here to obstruct the secondary catalytic mechanism in some 165 conformations ( Figure 6A ). In addition to the flexibility of the catalytic dyad, the size and flexibility of the 166 variable loop between the protease domain β1 and β2 strands and its interaction with the MTase-like 167 domain loop between β7 strand and α9 helix ( Figures 6B and 6C ) likely determine the rate of substrate 168 loading into the cleft and therefore cleavage kinetics. [52] Deletion of the exposed and most proximal 169 MTase-like domain residue (typically leucine, phenylalanine, or tryptophan) and sharper backbone 170 twisting by subsequent prolines in divergent alphaviruses may also widen the gap between these two 171 loops and affect substrate loading or may allow serine to better fit in the P1 pocket instead of the more 172 common alanine ( Figure 6D ). Sodium [53, 54] MTase-like domain and between its nsP3 macro and zinc-binding domains did not affect this distance. Nidogen-1 and nidogen-2 in healthy human cartilage and in late-525 stage osteoarthritis cartilage The expression of thrombospondin-4 528 correlates with disease severity in osteoarthritic knee cartilage Elastin-derived peptides 531 are involved in the process of human temporomandibular disorder by inducing inflammatory 532 responses in synovial cells The multifaceted roles of 535 perlecan in fibrosis Structural and genetic analysis of laminin-nidogen interaction Laminins in basement membrane assembly Muscular dystrophy meets protein biochemistry, the mother of invention A molecular understanding of alphavirus entry The α-macroglobulin bait region: Sequence diversity 546 and localization of cleavage sites for proteinases in five mammalian α-macroglobulins α2-macroglobulin bait region variants: A role for the bait region in 549 tetramer formation The regulation of translation in alphavirus-infected cells Cotranslational folding 554 stimulates programmed ribosomal frameshifting in the alphavirus structural polyprotein Involvement of human release 557 factors eRF3a and eRF3b in translation termination and regulation of the termination complex 558 formation The alphavirus capsid protein 563 inhibits IRAK1-dependent TLR signaling Interactions of alphavirus nsP3 protein with host proteins Alphavirus-induced hyperactivation of 568 PI3K/AKT directs pro-viral metabolic changes Connective tissue metabolism in chikungunya patients SH3 domain-mediated 573 recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication. PLOS 574 Pathog Structural basis of the high affinity 576 interaction between the Alphavirus nonstructural protein-3 (nsP3) and the SH3 domain of 577 amphiphysin-2 Viral resistance of MOGS-CDG patients implies a broad-spectrum strategy 580 against acute virus infection Evaluation of β1,4-galactosyltransferase in rheumatoid arthritis and its role in the 583 glycosylation network associated with this disease EDEM2 initiates mammalian 586 glycoprotein ERAD by catalyzing the first mannose trimming step Arms race between enveloped viruses and the host ERAD machinery Glucose trimming and mannose trimming affect 591 different phases of the maturation of Sindbis virus in infected BHK cells Morphogenesis of Sindbis virus in cultured Aedes albopictus cells Effect of N-acetylglucosamine 596 administration on cartilage metabolism and safety in healthy subjects without symptoms of arthritis: 597 A case report Glycan-dependent chikungunya viral infection 599 divulged by antiviral activity of NAG specific chi-like lectin Glucosamine promotes hepatitis B virus 602 replication through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 603 signaling O-linked N-acetylglucosamine glycosylation of 606 p65 aggravated the inflammation in both fibroblast-like synoviocytes stimulated by tumor necrosis 607 factor-α and mice with collagen induced arthritis Sumoylation 610 coordinates the repression of inflammatory and anti-viral gene-expression programs during innate 611 sensing The SUMOylation 613 pathway suppresses arbovirus replication in Aedes aegypti cells An expression 616 knowledgebase at single cell/nucleus level for the discovery of coding-noncoding RNA functional 617 interactions in skeletal muscle Human muscle satellite cells as 620 targets of chikungunya virus infection. PLOS One Infection of myofibers 623 contributes to increased pathogenicity during infection with an epidemic strain of chikungunya 624 virus Functional 626 processing and secretion of chikungunya virus E1 and E2 glycoproteins in insect cells Capsid-629 deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane Intercellular extensions are induced by the alphavirus structural 632 proteins and mediate virus transmission Chikungunya virus neutralization 635 antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants. PLOS 636 Pathog A picorna-like virus suppresses the N-end rule 640 pathway to inhibit apoptosis. eLife Life and death of proteins after protease cleavage: protein 642 degradation by the N-end rule pathway The STI1-domain is a flexible alpha-helical fold with a 647 hydrophobic groove Ubiquilins chaperone and 649 triage mitochondrial membrane proteins for degradation Immune deficiency, 652 autoimmune disease and intellectual disability: A pleiotropic caused by biallelic variants in the TPP2 653 gene A major role for 655 TPPII in trimming proteasomal degradation products for MHC class I antigen presentation Constitutive upregulation of the 658 transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts Dysregulated 661 TGF-β production underlies the age-related vulnerability to chikungunya virus Combined miRNA and 664 mRNA signature identifies key molecular players and pathways involved in chikungunya virus 665 infection in human cells BMP1 controls TGFβ1 activation via cleavage of latent TGFβ-binding 668 protein TGF-β and BMP signaling in osteoblast, skeletal development, and bone 672 formation, homeostasis and disease Opposite 675 effects of bone morphogenic protein-2 and transforming growth factor-β1 on osteoblast 676 differentiation TGF-beta1 on osteoimmunology and the bone component cells Arthritogenic alphaviral infection 680 perturbs osteoblast function and triggers pathologic bone loss Arthritogenic alphaviruses: new 683 insights into arthritis and bone pathology Interleukin 6, RANKL, and 686 osteoprotegerin expression by chikungunya virus-infected human osteoblasts Osteoclastogenesis induced by CHIKV-infected fibroblast-like synoviocytes: a possible interplay 690 between synoviocytes and monocytes/macrophages in CHIKV-induced arthralgia/arthritis Chikungunya virus infection impairs the function of osteogenic 693 cells. mSphere Identification 695 and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor 696 receptor 2 Pyrin-and CARD-only proteins as regulators of NLR functions Inhibition of 700 polyamine biosynthesis is a broad-spectrum strategy against RNA viruses Polyamines and their roles in virus infection Spermidine inhibits joints inflammation and macrophage 705 activation in mice with collagen-induced arthritis Spermidine restores dysregulated autophagy and 708 polyamine synthesis in aged and osteoarthritic chondrocytes via EP300 Spermidine activated RIP1 deubiquitination to 711 inhibit TNF-α-induced NF-κB/p65 signaling pathway in osteoarthritis Increased recycling of polyamines is associated with 714 global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum Increased polyamines may downregulate 717 interleukin 2 production in rheumatoid arthritis eIF5A promotes 720 translation of polyproline motifs eIF5A functions globally in translation 723 elongation and termination Polyamines enhance readthrough of the UGA termination 726 codon in a mammalian messenger RNA Histamine and histamine H4 receptor 729 promotes osteoclastogenesis in rheumatoid arthritis. Sci Rep Bioactive lipids and chronic inflammation: managing the 732 fire within The glutathione defense system in the 734 pathogenesis of rheumatoid arthritis Alphavirus-induced membrane rearrangements during replication, 737 assembly, and budding. Pathogens Spatial and temporal analysis of alphavirus replication and assembly 740 in mammalian and mosquito cells