Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C1175175 (SARS)
19,188 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a promising avenue to design specific inhibitors for 3CL proteases by disrupting their dynamic correlation network.
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PMID:Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain. 2503 52

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.
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PMID:Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes. 2507 27

The spike (S) protein of severe acute respiratory syndrome-associated CoV (SARS-CoV) mediates membrane fusion and viral entry. These events involve structural rearrangements, including heteromerization between two heptad repeats (HR1 and HR2) to form a trimer of dimers as a six-helix bundle (6-HB), a quaternary protein structure that brings two distant clusters of hydrophobic sequences into the proximity of each other, the internal fusion peptide (IFP) preceding HR1, and the highly conserved tryptophan (Trp)-rich membrane proximal external region (MPER) following HR2. Here, we show that MPER can undergo self-oligomerization and heteromerization with IFP, events that are Trp-dependent. To delineate the roles of Trp residues of MPER in forming these quaternary structures and interacting with membranes, we employed a panel of synthetic peptides: MPER peptide (M-wt) and its alanine (Ala) and phenylalanine (Phe) analogues. Ala substitutions of Trp inhibited its association with cellular membranes. Chemical cross-linking experiments showed that M-wt can self-interact to form oligomers and cross-interact with IFP23, a synthetic IFP peptide, to form a heterohexamer. In comparison, little high-order oligomer was formed between M-wt and fusion peptide. The specific interaction between M-wt and IFP23 was confirmed by immunofluorescence staining experiments. In aqueous solutions, both M-wt and IFP23 displayed random secondary structures that became helical in hydrophobic solvents. Triple-Ala substitutions of Trp in M-wt, but not the corresponding triple-Phe analogue, disrupted oligomerization of M-wt and hetero-oligomerization of M-wt with IFP23. Overall, our results show that Trp residues of MPER play a key role in maintaining the structure and functions of MPER, allowing it to interact with IFP to form a MPER-IFP heteromer, a putative quaternary structure extending from the 6-HB, and function in membrane fusion. Finally, we showed that a MPER peptide could serve as an inhibitor in the entry process.
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PMID:Tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of SARS-CoV spike protein. 2566 3

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.
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PMID:A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex. 2614 21

RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.
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PMID:Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses. 2630 38

Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a C-type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC-SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand-off to another receptor or a co-receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild-type DC-SIGN or 2 internalization-deficient DC-SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super-resolution imaging and high content single particle tracking, we investigated DENV binding, DC-SIGN surface transport, endocytosis, as well as cell infectivity. DC-SIGN was found colocalized with DENV inside cells suggesting hand-off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC-SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co-receptor because cells expressing the internalization-deficient mutants could still be infected.
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PMID:Beyond attachment: Roles of DC-SIGN in dengue virus infection. 2812 92

The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, ⁴⁷⁵NVCWAK⁴⁸⁰, which differs from that of papain (²²CGSCWAFS²⁹). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state in which the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed-consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in k_cat/K_m. A side chain-substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in K_m. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.
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PMID:Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State. 2906 79

The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3'-to-5' exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.
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PMID:Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations. 2911 26

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.
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PMID:Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses. 2926 63

The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.IMPORTANCE Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.
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PMID:Fitness Barriers Limit Reversion of a Proofreading-Deficient Coronavirus. 3134 Oct 46


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