Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.     

African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae

We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition.     

Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.     

Viral proteins expressed on the surface of murine leukemia cells.

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.     

The role of the S-S bridge in retroviral protease function and virion maturation

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.     

Selection of Functional Variants of the NS3-NS4A Protease of Hepatitis C Virus by Using Chimeric Sindbis Viruses

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417–1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.     

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.     

Retroviral proteases

SUMMARY: The proteases of retroviruses, such as leukemia viruses, immunodeficiency viruses (including the human immunodeficiency virus, HIV), infectious anemia viruses, and mammary tumor viruses, form a family with the proteases encoded by several retrotransposons in Drosophila and yeast and endogenous viral sequences in primates. Retroviral proteases are key enzymes in viral propagation and are initially synthesized with other viral proteins as polyprotein precursors that are subsequently cleaved by the viral protease activity at specific sites to produce mature, functional units. Active retroviral proteases are homodimers, with each dimer structurally related to the larger class of single-chain aspartic peptidases. Each monomer has four structural elements: two distinct hairpin loops, a wide loop containing the catalytic aspartic acid and an alpha helix. Retroviral gene sequences can vary between infected individuals, and mutations affecting the binding cleft of the protease or the substrate cleavage sites can alter the response of the virus to therapeutic drugs. The need to develop new drugs against HIV will continue to be, to a large extent, the driving force behind further characterization of retroviral proteases.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.     

N-Terminal Modifications of Ubiquitin via Methionine Excision,Deamination, and Arginylation Expand the Ubiquitin Code

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.     

Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 Å, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU''s preference for Ub and Ub-like substrates.Crimean-Congo hemorrhagic fever (CCHF) is characterized in humans by the sudden onset of fever, myalgia, headache, dizziness, sore eyes, photophobia, and hyperanemia as well as severe hemorrhages (28, 43, 46). The causative agent of CCHF is the CCHF virus, which is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] virus of the genus Nairovirus, belonging to the viral family Bunyaviridae. Originally named after outbreaks in the former Soviet Union and in the Congo during the mid-20th century, the affected area of this disease has rapidly spread to large areas of sub-Saharan Africa, the Balkans, Northern Greece, European Russia, Pakistan, the Arabian Peninsula, Iran, Afghanistan, Iraq, Turkey, and recently, the Xinjiang province of China (43, 46). The CCHF viral genome, as well as those of the closely related Dugbe and Nairobi viruses, consists of three negative-sense RNA segments, small (S), medium (M), and large (L). Incubation of CCHF is 5 to 6 days, with fatalities occurring less than 7 days after signs of infection. Fatality rates for patients infected with the CCHF virus ranged from 5% to 70%, depending on phylogenetic variation of the virus, transmission route, treatment facility, and the reporting and confirmation of the case statistics (19, 32, 43, 47).The innate immune system serves as the human''s first line of defense from invading pathogens, including CCHF virus. The type I interferon (IFN) response comprises a key component of this system by upregulating more than 300 IFN-stimulated genes (ISGs) whose products detect viral molecules, promote amplification of the type I IFN response, modulate other signaling pathways, and directly provide antiviral activity (34). Regulation of the type I IFN response has been shown to rely on posttranslational modification by ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15) (14, 23). Both Ub and ISG15 are expressed in a proform and cleaved to leave a double-glycine C terminus that forms an isopeptide bond with predominantly the ɛ-NH₂ of lysine residues of a target protein through a three-step enzymatic process. In addition to forming isopeptide bonds with target proteins, Ub, which contains seven lysine residues, has been observed to form poly-Ub chains. The most studied of these moieties are K29-linked, K48-linked, and K63-linked poly-Ub. While K29-linked and K48-linked polyubiquitination of proteins leads to their degradation in the lysosome and proteasome, respectively, conjugation of K63-linked poly-Ub to proteins has an activating effect, resulting in an enhanced type I IFN response (2, 7, 18, 33, 40). Currently, more than 150 proteins have been identified as forming conjugates with ISG15, with the number of proteins forming Ub conjugates far exceeding that number (12, 48). A subset of type I IFN signaling and effector proteins that Ub and ISG15 have been shown to stabilize includes JAK1, STAT1/2, double-stranded RNA-dependent protein kinase (PKR), myxovirus-resistant protein A (MxA), and RIG-I (17). MxA has particularly shown to be important in type I IFN response to CCHF infection. RIG-I and several other proteins have also been shown to be targets for K63-linked poly-Ub (4).Recently, investigators have identified a cysteine viral ovarian tumor domain (vOTU) protease colocated with the RNA-dependent RNA polymerase in the L protein of the CCHF virus (14). Interestingly, as CCHF is an ssRNA(−) virus, no protease is required to cleave a viral polypeptide to facilitate viral replication as in positive-sense ssRNA [ssRNA(+)] viruses. Furthermore, recent reports have observed that vOTU is not required for RNA-dependent RNA polymerase activity and for vOTU protease activity linked to impairment of the type I IFN response through its deubiquitinating and deISGylating activity (6, 14). Additional studies have also tentatively identified the presence of vOTU homologues in the Arterivirus genus of the Arteriviridae family, suggesting that they too may facilitate impairment of the type I IFN response (14). Since the discovery of the first ovarian tumor domain (OTU) protease in Drosophila oogenesis and prior to the identification of vOTU, OTU superfamily members could be divided into three subclasses according to their sequence homology, otubains, A20-like OTUs, and ubiquitin thioesterase ZRANB1 (22). With the addition of the viral OTU subclass, OTU superfamily members in more than 100 eukaryotic, bacterial, and viral proteins have now been identified (6, 27). Predominantly, OTU proteases have been linked to ubiquitin (Ub) removal and/or remodeling of Ub-conjugated proteins, placing them among five protease superfamilies that facilitate signal transduction cascades and play key roles in protein stability (22). However, vOTU is unique in that it is the only OTU to have shown both deubiquitinating and deISGylating activity (14). Instead, Otubain1/2 (OTUB1/2) plays a key role in T cell response and prefers K48-linked poly-Ub or NEDD8 (neural precursor cell expressed, developmentally downregulated 8) as a substrate (12). A20 and A20-like Cezanne OTU proteases are negative regulators of the NF-κB-mediated inflammation response, selectively cleaving K63-linked poly-Ub targets. DUBA also shows preference for K63-linked poly-Ub (20). In attempts to better understand the OTU superfamily, structures of OTUB and A20-like OTU domains have been elucidated (12, 21, 30). An X-ray structure of the yeast ovarian tumor 1 (yOTU1) domain, which interacts with Cdc48 and has a preference for K48-linked poly-Ub, was achieved in complex with mono-Ub (27). However, since yOTU1 has a preference for K48-linked Ub and possesses low sequence identity to vOTU and other OTU domain proteases, only limited information on vOTU could be obtained. In addition to vOTU, several other viral proteases, such as papain-like protease (PLpro) from the severe acute respiratory syndrome (SARS) coronavirus, have also shown deubiquitinating and deISGylating activity to evade the innate immune system (6, 8, 43, 49). However, no viral proteases that are known to possess deISGylating activity have been visualized as being bound to Ub or Ub-like substrates. To address this issue and elucidate the atomic-level structure of a member from the viral OTU superfamily subclass, we have obtained the X-ray crystal structure of vOTU bound with Ub (vOTU-Ub). We also have characterized the vOTU substrate specificity for mono-Ub, ISG15, and NEDD8 and compared the results with those from human OTUB2 (hOTUB2). Additionally, we assessed vOTU''s deubiquitinating activity toward K48- and K63-linked poly-Ub.     

Structural and biochemical characterization of SADS‐CoV papain‐like protease 2

Swine acute diarrhea syndrome coronavirus (SADS‐CoV) is a novel coronavirus that is involved in severe diarrhea disease in piglets, causing considerable agricultural and economic loss in China. The emergence of this new coronavirus increases the importance of understanding SADS‐CoV as well as antivirals. Coronaviral proteases, including main proteases and papain‐like proteases (PLP), are attractive antiviral targets because of their essential roles in polyprotein processing and thus viral maturation. Here, we describe the biochemical and structural identification of recombinant SADS papain‐like protease 2 (PLP2) domain of nsp3. The SADS‐CoV PLP2 was shown to cleave nsp1 proteins and also peptides mimicking the nsp2|nsp3 cleavage site and also had deubiquitinating and deISGynating activity by in vitro assays. The crystal structure adopts an architecture resembling that of PLPs from other coronaviruses. We characterize both conserved and unique structural features likely directing the interaction of PLP2 with the substrates, including the tentative mapping of active site and other essential residues. These results provide a foundation for understanding the molecular basis of coronaviral PLPs' catalytic mechanism and for the screening and design of therapeutics to combat infection by SADS coronavirus.     

Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition.

The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.     

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.     

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.