In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the ...genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.
•The genomic sequence of 2019-nCoV indicates that the virus clusters with betacoronaviruses of lineage b.•2019-nCoV S-protein sequence has a specific furin-like cleavage site absent in lineage b CoV including SARS-CoV sequences.•The furin-like cleavage site in the S-protein of 2019-nCoV may have implications for the viral life cycle and pathogenicity.•Campaigns to develop anti-2019-nCoV therapeutics should include the evaluation of furin inhibitors.
Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a ...diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.
The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a ...novel class of small molecules (1,2,3triazolo4,5-dpyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly ...broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4–8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
Display omitted
•We developed a highly sensitive cell-based Nanoluciferase assay to screen for inhibitors of SARS-CoV-2 main protease.•We provide evidence that the assay is scalable to the 384-well format and robust (z’ factor of 0.8).•The false positive rate is minimized: gain of signal assay, catalytically inactive protease control.•The assay can be adapted to other coronaviruses and viruses which rely on a viral protease.•We report the identification of 2 lead compounds that inhibit nsp5 activity and SARS-CoV-2 replication in cell culture.
Several lines of evidence indicate that HIV infection can result in several possible incomes, including a very small proportion of individuals whose HIV replication is controlled after treatment ...interruption (known as HIV posttreatment controllers) or spontaneously without any treatment (known as HIV elite controllers). Both types of individuals are HIV RNA negative but HIV DNA positive, with living virus which can be stimulated ex vivo. A review was conducted to assess the literature on yet rarer cases with detectable integrated HIV DNA without HIV infectious virus in HIV-seropositive or -negative individuals. Three categories of patients were identified: (a) HIV-seropositive individuals with apparent spontaneous cure from their HIV infection, (b) HIV-seronegative children born to HIV-infected mothers and (c) highly exposed seronegative adults. Validity criteria were proposed to assess the presence of integrated HIV DNA as possible or unquestionable in these three categories. Only three articles among the 22 ultimately selected fulfilled these criteria. Among the highly exposed seronegative subjects, some individuals were described as being without integrated HIV DNA, probably because these subjects were not investigated using relevant, highly sensitive methods. Finally, we propose a definition of spontaneous cure of HIV infection based on clinical, immunologic and virologic criteria.
•Production of two 74-mer capped RNA substrates, GpppA2′OMe-RNA74 and 7MeGpppA-RNA74, for DENV N7- and 2′-O-MTase.•Development of a robust, specific and high-throughput N7-MTase inhibition ...assay.•Selective screening of known MTase inhibitors against DENV 2′-O- and N7-MTase.
Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, 7MeGpppA2′OMe-RNA, at the 5′-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, 7MeGpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA2′OMe-RNA74, by ligation of two RNA fragments. Then, we use GpppA2′OMe-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate 7MeGpppA-RNA74 in order to characterize the DENV 2′-O-MTase activity specifically on long capped RNA.
We next compared the N7- and 2′-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.
The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase
DV, is involved in two of four steps required for the formation of the viral mRNA cap
7MeGpppA
...2′OMe, the guanine-N7 and the adenosine-2′O methylation. Its
S-adenosyl-
l-methionine (AdoMet) dependent 2′O-methyltransferase (MTase) activity has been shown on capped
7Me±GpppAC
n
RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 Å to 2.8 Å resolution of NS5MTase
DV in complex with cap analogues and the co-product of methylation
S-adenosyl-
l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP–binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of
7MeGpppAC
4 and
7MeGpppAC
5 RNAs is inhibited in the presence of GTP,
7MeGTP and
7MeGpppA but not by ATP. This particular position of the cap is in accordance with the 2′
O-methylation step. A model was generated of a ternary 2′
O-methylation complex of NS5MTase
DV,
7MeGpppA and AdoMet. RNA-binding increased when
7Me±GpppAGC
n-1
starting with the consensus sequence GpppAG, was used instead of
7Me±GpppAC
n
. In the NS5MTase
DV–GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5′ end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2′O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative
Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, 7MeG5′-ppp5′-A2′OMe. The positive-sense RNA genomes of flaviviruses ...(Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and 7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC
n
and 7MeGpppAC
n
(1 ≤ n ≤ 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 ≤ n ≤ 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
PDF
