Expanding Repertoire of Plant Positive-Strand RNA Virus Proteases

Abstract

Many plant viruses express their proteins through a polyprotein strategy, requiring the acquisition of protease domains to regulate the release of functional mature proteins and/or intermediate polyproteins. Positive-strand RNA viruses constitute the vast majority of plant viruses and they are diverse in their genomic organization and protein expression strategies. Until recently, proteases encoded by positive-strand RNA viruses were described as belonging to two categories: (1) chymotrypsin-like cysteine and serine proteases and (2) papain-like cysteine protease. However, the functional characterization of plant virus cysteine and serine proteases has highlighted their diversity in terms of biological activities, cleavage site specificities, regulatory mechanisms, and three-dimensional structures. The recent discovery of a plant picorna-like virus glutamic protease with possible structural similarities with fungal and bacterial glutamic proteases also revealed new unexpected sources of protease domains. We discuss the variety of plant positive-strand RNA virus protease domains. We also highlight possible evolution scenarios of these viral proteases, including evidence for the exchange of protease domains amongst unrelated viruses.

Keywords: protease specificity; protease structure; proteolytic processing; viral proteases; virus evolution.

Figure 1

Chymotrypsin-like cysteine or serine proteases. (A) Genomic organization of representative viruses. Polyproteins are shown by boxes with cleavage sites indicated by vertical lines. Protease domains are shown in red for chymotrypsin-like 3C or 3CL cysteine proteases, brown for chymotrypsin-like serine proteases, green for papain-like cysteine proteases, and blue for glutamic proteases. The two shades of brown in the polyprotein of potyvirids represent P1 serine proteases of type A (light brown) or type B (dark brown). The same color code is used for arrows above each cleavage site indicating the protease responsible for the cleavage. A black arrow in the PV polyprotein indicates an autocatalytic maturation cleavage event of the capsid protein. Orange boxes indicate the VPg proteins. Purple ovals represent the conserved picorna-like RdRp domain. Stars indicate coat protein domains: purple for the three picorna-like type 1 jelly-roll domains (which can be divided into one, two, or three CPs, depending on the genera), pink for type 2 jelly-roll domains, and blue for CPs forming filamentous virions. Helicase domains are shown by the triangles: purple for superfamily 3 helicases and pink for superfamily 2 helicases. The protease co-factor motif of CPMV is shown by a small green square. This motif is present in nepoviruses (represented by the empty green square) but does not act as a co-factor. (B) Structure of representative proteases. Catalytic residues are represented as follows. Cellular protease: chymotrypsin A from Bos taurus (pdb:1CBW_ABC) His⁵⁷ (purple), Asp¹⁰² (pink), Ser¹⁹⁵ (orange), human virus protease: PV 3C-Pro (pdb: 1L1N_A): His¹⁶⁰³ (purple), Glu¹⁶³⁴ (blue), Cys¹⁷¹⁰ (yellow) and plant virus proteases: TEV NIa 3CL-Pro (pdb: 1LVM): His²⁰⁸³ (purple), Asp²¹¹⁸ (pink), Cys²¹⁸⁸ (yellow), and SeMV serine Pro (pdb: 1ZYO): His¹⁸¹ (purple), Asp²¹⁶ (pink), Ser²⁸⁴ (orange). Images of protease structures are reprinted with permission from the MEROPS database (www.ebi.ac.uk/merops). PV: poliovirus, CPMV: cowpea mosaic virus, GFLV: grapevine fanleaf virus, ToRSV: tomato ringspot virus, SMoV: strawberry mottle virus, TEV: tobacco etch virus, CVYV: cucumber vein yellowing virus, UCBSV: Ugandan cassava brown streak virus, SeMV: sesbania mosaic virus.

Figure 2

Papain-like cysteine proteases. (A) Genomic organization and proteolytic cleavages of representative viruses. Polyproteins are shown by boxes with cleavage sites indicated by vertical lines. Protease domains are shown in green for papain-like cysteine proteases, red for chymotrypsin-like 3CL cysteine proteases and brown for chymotrypsin-like serine proteases. The same color code is used for arrows above each cleavage site indicating the protease responsible for the cleavage. A black arrow in the foot-and-mouth disease virus (FMDV) polyprotein indicates an autocatalytic maturation cleavage event of the capsid protein. The “ngpg” sequence represent the 2A translational stop-go sequence of FMDV. Orange boxes indicate the VPg proteins. Ovals represent RdRp domains: purple for picorna-like RdRp and blue for alpha-like RdRp. Stars indicate the coat protein domains: purple for picorna-like type 1 jelly-roll domains icosahedral coat protein, red for unrelated icosahedral CP of togaviruses, blue for filamentous coat proteins. Helicase domains are shown by the triangles: light blue, pink and purple for helicase superfamilies 1, 2, and 3, respectively. Please note that cleavage sites processed only by viral proteases are shown. The VEEV cleavage sites processed by the cellular furin protease are not shown. (B) Structure of representative proteases. Catalytic residues are represented, as follows; Cellular protease: papain from Carica papaya (pdb: 1PE6, in complex with E-64 inhibitor, which is shown in grey): Cys¹⁵⁸ (yellow), His²⁹² (purple), Asn³⁰⁸ (pink), animal virus proteases: FMDV L-Pro (pdb: 1QMY_A: mutant protease with Cys⁵¹ mutated to Ala), Cys⁵¹ (yellow, mut. to Ala), His¹⁴⁸ (purple), Asp¹⁶³ (pink), and VEEV nsP2 protease (pdb: 2HWK, only the protease catalytic domain is shown): Cys⁴⁷⁷ (yellow), His⁵⁴⁶ (purple), and plant virus protease: TuMV HC-Pro (pdb: 3RNV, protease catalytic domain): Cys⁷⁰⁶ (yellow), His⁷⁷⁹ (purple). Images of protein structures are reprinted with permission from the MEROPS database (www.ebi.ac.uk/merops). FMDV: foot-and-mouth disease virus, VEEV: Venezuelan equine encephalitis virus, TEV: tobacco etch virus, CYNMV: Chinese yam necrotic mosaic virus, BaYMV: barley yellow mosaic virus, BYV: beet yellows virus, CTV: citrus tristeza virus, TuMV: turnip mosaic virus.

Figure 3

Ovarian Tumor Domain (OTU)-like cysteine proteases. (A) Genomic organization and proteolytic cleavages of representative (+)-strand RNA viruses. Polyproteins are shown by boxes with cleavage sites indicated by vertical lines. Protease domains are shown in dark green for papain-like cysteine proteases, lime green for OTU-like cysteine proteases and brown for chymotrypsin-like serine proteases. The same color code is used for arrows above each cleavage site indicating the protease responsible for the cleavage. The yellow square represents an OTU-like domain that does not orchestrate viral polyprotein cleavage. Ovals represent RdRp domains: purple for picorna-like RdRp and blue for alpha-like RdRp. Stars indicate the coat protein domains: green for the arteriviridae nucleocapsid, pink for type 2 jelly-roll domains and blue for filamentous coat proteins. Helicase domains are shown by the triangles: light blue for superfamily 1. (B) Structure of representative deubiquitinases (DUB) and/or proteases. Catalytic residues are represented as follows: Yeast OTU DUB (pdb: 3C0R, in complex with ubiquitin, only one monomer of the trimer is shown): Asp¹⁷⁷ (pink), Cys¹²⁰ (yellow), and His²²² (purple). Animal negative-strand RNA virus DUB: CCHMV OTU DUB (pdb: 3PT2_A): Cys⁴⁰ (yellow), His¹⁵¹ (purple), and Asp¹⁵³ (pink). (Image of the yeast and CCHMV OTU DUB structures are reprinted with permission from the MEROPS database (www.ebi.ac.uk/merops)). Animal (+)-strand RNA virus protease-DUB: EAV nsP2 protease (pdb: 4IUM) Cys²⁷⁰ (yellow) and His³³² (purple) and plant (+)-strand RNA virus protease-DUB: TYMV OTU-like protease (pdb: 4A5U): Cys⁷⁸³ (yellow) and His⁸⁶⁹ (purple). Image for the EAV and TYMV protease structure were generated using PyMol. EAV: equine arteritis virus, TYMV: turnip yellow mosaic virus, BBScV: blueberry scorch virus, PVX: potato virus X, CCHMV: Crimean-Congo hemorrhagic fever virus.

Figure 4

Confirmed and putative glutamic proteases. (A) Genomic organization and proteolytic cleavages of representative viruses. Polyproteins are shown by boxes with cleavage sites indicated by vertical lines. Protease domains are shown in green for papain-like cysteine proteases, red for chymotrypsin-like 3CL cysteine proteases, and blue for confirmed (SMoV) or putative (TICV) glutamic protease domain. The same color code is used for arrows above each cleavage site indicating the protease responsible for the cleavage. The orange box indicates the VPg protein. Ovals represent RdRp domains: purple for picorna-like RdRp and blue for alpha-like RdRp. Stars indicate the coat protein domains: purple for picorna-like type 1 jelly-roll domains icosahedral coat protein and blue for filamentous coat proteins. Helicase domains are shown by the triangles: light blue and purple for helicase superfamilies 1 and 3, respectively. (B) Structure of representative proteases. Catalytic residues are represented as follows: Fungal protease: Scyatalidium lignicolum glutamic peptidase (pdb: 1S2B): Gln¹⁰⁷ (pink) and Glu¹⁹⁰ (blue) (Image reprinted with permission from the MEROPS database (www.ebi.ac.uk/merops)) and plant virus protease: SMoV Pro2-Glu putative model of the catalytic region, generated using Phyre2 [35]: Glu¹¹⁹² and Glu¹²⁷⁴ (blue) and Gln¹¹⁸⁰ (pink). Image of the structure model was generated using PyMol. SMoV: strawberry mottle virus, TICV: tomato infectious chlorosis virus.