A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.

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.

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.

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 high-throughput 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 four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC 50 values in the 4-21 µM range. The here described 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.

Nucleoside and nucleotide inhibitors of HIV-1 replication.

HIV-1 reverse transcriptase (RT) is one of the main targets for antiviral therapy. Two classes of RT inhibitors can be distinguished: those that are nucleoside or nucleotide analogues (sharing the common NRTIs abbreviation) and those that are not. This review focuses on the NRTIs, which are highly efficient in slowing down viral replication and are used in combination regimens. Unfortunately, the current inhibitors do not completely suppress viral replication and due to the high capacity of adaptation of HIV, allow the selection of drug-resistant viruses. Resistance mechanisms to NRTIs have been extensively investigated and can be divided into two types: improved discrimination of a nucleotide analogue relative to the natural substrate or increased phosphorolytic cleavage of an analogue-blocked primer. This knowledge is important both for the development of new drugs designed to target resistant strains and for the development of new antiviral strategies. The NRTIs currently in clinical trials and new developments in this area are also reviewed.

The emergence of different resistance mechanisms toward nucleoside inhibitors is explained by the properties of the wild type HIV-1 reverse transcriptase.

Nucleoside reverse transcriptase inhibitors (NRTIs) represent one of the main drug families used against AIDS. Once incorporated in DNA, they act as chain terminators, due to the lack of a 3'-hydroxyl group. As for the other anti-human immunodeficiency virus type 1 drugs, their efficiency is limited by the emergence of resistant viral strains. Unexpectedly, previous studies indicated that resistance toward NRTIs is achieved via two distinct and generally exclusive mechanisms. Resistance mutations either decrease the efficiency of NRTIs incorporation or increase their excision from the extended primer. To understand the emergence of different resistance mechanisms toward a single inhibitor class, we compared the incorporation and the pyrophosphorolysis of several NRTIs using wild type reverse transcriptase (WT RT). We found that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway. Indeed, our results suggest that the pathway by which RT become resistant toward a given NRTI can be predicted by studying the inhibition of WT RT, because the resistance mutations do not confer new properties to the mutant enzyme, but rather exacerbate pre-existing properties of the WT enzyme. They also help to understand the low cross-resistance toward d4T observed with the 3'-azido-3'-deoxythymidine (AZT or zidovudine)-resistant RT.

Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis.

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.

Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.

The human immunodeficiency virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the pre-initiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo*. Formation of the RNA polymerase IIo* occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-beta- D -ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA.

Structural basis for the specificity of the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV-1 RNA and tRNA3Lys, but remains poorly understood at the three-dimensional level. We used chemical probing to gain insight into the three-dimensional structure of the viral RNA-tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three-dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA-tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3' end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure.

Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex.

Retroviruses use a specific tRNA, whose 3' end is complementary to the 18 nucleotides of the primer binding site (PBS), to prime reverse transcription. Previous work has shown that initiation of HIV-1 reverse transcription is a specific process, in contrast with the subsequent elongation phase. HIV-1 reverse transcriptase (RT) specifically recognizes the complex formed by the viral RNA and tRNA3Lys. We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing. In this model, tRNA3Lysextensively interacts with the genomic RNA. Here, we have combined site-directed mutagenesis and structural probing to test crucial aspects of this model. We found that the complex interactions between tRNA3Lysand HIV-1 RNA, and the intra-molecular rearrangements did not depend on the presence of upstream and downstream viral sequences. Indeed, a short RNA template, encompassing nucleotides 123-217 of the HIV-1 Mal genome, was able, together with the primer tRNA, to adopt the same structure as longer viral RNA fragments. This model primer/template is thus amenable to detailed structural and functional studies. The probing data obtained on the tRNA3Lys/mutant viral RNA complexes support the previously proposed model. Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA3Lysand the so-called viral 'A-rich loop' destabilizes all four helices of the extended tRNA3Lys/HIV-1 RNA interactions.

Two step synthesis of (-) strong-stop DNA by avian and murine reverse transcriptases in vitro.

Retroviral reverses transcriptases (RTs) are RNA- and DNA-dependent DNA polymerases that use a tRNA bound at the so-called primer binding site (PBS) located near the 5'end of the genomic RNA as primer. Thus, RTs must be able to accommodate both RNA and DNA in the primer strand. To test whether the natural primer confers some advantages to the priming process, we compared initiation of reverse transcription of avian and murine retroviral RNAs, using either their natural tRNA primer, tRNATrp and tRNAPro, respectively, or synthetic 18mer oligodeoxyribonucleotides (ODNs) and oligoribonucleotides (ORNs) complementary to their PBS. In both retroviral systems, the initial extension of ODNs was fast and processive. The initial extension of ORNs, tRNATrp and tRNAPro was much slower and distributive, giving rise to the transient accumulation of short pausing products. Synthesis of (-) strong-stop DNA was delayed when using ORNs and tRNAs, compared to ODNs. Even though ORNs and tRNAs were initially extended at the same rate, the short pausing products were more rapidly extended when using the tRNA primers. As a consequence, synthesis of (-) strong-stop DNA was much more efficient with tRNA primers, compared to ORNs. Taken together, these results suggest that the tRNA-primed synthesis of (-) strong-stop DNA is a two-step process, as already observed for HIV-1. The initiation mode corresponds to the initial non-processive nucleotide addition and extension of the short pausing products. It is more efficient with the natural primers than with ORNs. Initiation is followed by a more processive and unspecific elongation mode. Elongation is observed when the primer strand is DNA, i.e. when using the ODNs as primers or when the ORN and tRNA primers have been extended by a sufficient number (depending on the retroviral system) of deoxyribonucleotides.

Probing the higher order structure of RNA with peroxonitrous acid.

Potassium peroxonitrite (ONOOK) and [Fe(EDTA)]2- were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]2- gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOH) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]2- is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the 'inside' of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA(Lys3) which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA(Lys3).

Determining the conformation of RNAs in solution. Application to a retroviral system: structure of the HIV-1 primer binding site region and effect of tRNA(3Lys) binding.

RNAs play a crucial and central role in a large variety of biological functions obviously linked to the wide variety of structures that they can adopt. Understanding the function of RNAs thus requires the knowledge of their two- and three-dimensional structures. We describe in detail the way to access the secondary structure of RNAs, by combining sequence comparison, secondary structure prediction by computer and, mainly, experimental data obtained by probing with chemicals and ribonucleases. These approaches were used to investigate secondary structure of the region containing the primer binding site of HIV-1 genomic RNA either free or involved in the binary complex with the replication primer tRNA(3Lys).

Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys).

Initiation of reverse transcription is a crucial step of retroviral infection. In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA3(Lys) to the primer binding site (PBS) of the viral RNA. Moreover, additional interactions between the two RNAs were recently evidenced [Isel et al. (1995) J. Mol. Biol. 247, 25269-25272]. To get further information on the topology of the viral RNA/tRNA3(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained. The crosslinked regions were characterized by RNase T1 digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm5S2U34 and U35 in the anticodon loop of tRNA3(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis. The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA. Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.

Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys.

Initiation of RNA-dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV-1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV-1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo- and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV-1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV-1 RNA, HIV-1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer-template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.

Structural and functional evidence that initiation and elongation of HIV-1 reverse transcription are distinct processes.

Retroviral reverse transcription starts with the extension of a cellular tRNA primer bound near the 5' end of the viral genomic RNA at a site called the primer binding site (PBS). Formation of the HIV-1 initiation complex between tRNA3(Lys), viral RNA and reverse transcriptase probably occurs during encapsidation of these components. tRNA3(Lys) is thought to be selectively packaged by interaction with the reverse transcriptase domain of the Pr160Gag-Pol precursor protein, then annealed to the PBS of viral RNA with the help of the nucleocapsid protein. tRNA3(Lys) and HIV-1 viral RNA form a highly-structured complex, with extended interactions between the two molecules. Two different modes of reverse transcription have been distinguished: initiation, a tRNA3(Lys)-specific and distributive mode of polymerization corresponding to the addition of the first five nucleotides, followed by elongation, a non-specific and processive mode of DNA synthesis. These two modes are reminiscent of the initiation and elongation processes previously observed with DNA-dependent RNA polymerases.

Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer).

Reverse transcription of human immunodeficiency virus type-1 (HIV-1) genomic RNA is primed by tRNA(3Lys), whose 3' end 18 nucleotides are complementary to the viral primer binding site (PBS). We used chemical and enzymatic probes to test the conformation of the viral RNA and tRNA(3Lys), in their free form and in the HIV-1 RNA/tRNA(3Lys) binary complex. Extensive reactivity changes were observed in both molecules upon formation of the binary complex. In the viral RNA, reactivity changes occurred up to 69 nucleotides upstream and 72 nucleotides downstream of the PBS. A secondary structure model of the HIV-1 RNA/tRNA(3Lys) complex accounting for all probing data has been constructed. It reveals an unexpectedly complex and compact pseudoknot-like structure in which most of the anticodon loop, the 3' strand of the anticodon stem and the 5' part of the variable loop of tRNA(3Lys) interact with viral sequences 12 to 39 nucleotides upstream of the PBS. The core of the binary complex is a complex junction formed by two single-stranded sequences of tRNA(3Lys), an intramolecular viral helix, an intramolecular tRNA helix, and two intermolecular helices formed by the template/primer interaction. This junction probably highly constrains the tertiary structure of the HIV-1 RNA/tRNA(3Lys) complex. Compared to the structure of the free molecules, only the D arm of tRNA(3Lys) and a small viral stem-loop downstream of the PBS are unaffected in the binary complex. Sequence comparison reveals that the main characteristics of the binary complex model are conserved among all HIV-1 isolates.

tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription.

In all retroviruses, reverse transcription is primed by a tRNA whose 3' end 18 nucleotides are complementary to the so called viral primer binding site. Previous work showed that reverse transcription of HIV-1 RNA is initiated by tRNA(3Lys). Using a variety of chemical and enzymatic structural probes, we investigated the interactions between HIV-1 RNA and its natural primer tRNA(3Lys). In addition to the predictable contacts between the viral primer binding site and the 3' end of tRNA(3Lys), a specific interaction takes place between an A-rich loop located upstream of the primer binding site region and the anticodon loop of tRNA(3Lys). This AAAA/Umcm5s2UUU loop-loop interaction is not observed when the natural primer is replaced by an in vitro synthesized tRNA(3Lys) transcript. Furthermore, dethiolation of the modified nucleotide mcm5s2U at position 34 of tRNA(3Lys) strongly destabilizes this interaction. Sequence and structure comparisons indicate that the primer/template loop-loop interaction is conserved in all HIV-1 isolates, and possibly also in HIV-2 and SIV.

Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains.

The 5' region of HIV-1 RNA contains functional elements involved in key steps of the retroviral cycle, such as genomic RNA transcription, splicing, translation, dimerization or initiation of reverse transcription. In the present work, we investigated the conformation of the first 500 nucleotides covering the RNA leader and the 5' gag coding sequences of HIV-MAL, using chemical probing. We provide detailed information on almost each nucleotide at one of their Watson-Crick positions and on position N-7 of purines. Experiments were conducted on two in vitro transcribed RNA fragments (1 to 707 and 1 to 311). A secondary structure model was derived by combining the experimental data, computer predictions and sequence comparison. Under conditions favoring dimerization (high salt concentration), HIV-1 RNA folds into independent structural domains that can be related to defined functional regions. The first domain corresponds to TAR forming a stable stem-loop. Intrinsic structural features are found to stabilize the TAR hairpin loop. The second domain (nucleotides 56 to 299) contains the PBS sequence, which is located in a stable subdomain constrained by a four stem junction (nucleotides 139 to 218). Although the MAL isolate has an insertion near the PBS, probably resulting from the duplication of a 23-nucleotide sequence, the structural organization of this subdomain is conserved in all other HIV-1 isolates. The third domain (nucleotides 300 to 404) contains the splice donor site, packaging and dimerization elements and the AUG initiation codon of gag. A major result is the structural versatility of this region. Two mutually exclusive structures, both equally in agreement with probing data, could modulate the different functions involving this domain. The reduced accessibility of the gag translational initiation site possibly accounts for the low efficiency of the in vitro translation of the dimer. Finally, the 5' gag coding sequences form a metastable domain.