Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 × 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for future development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

An unexpected, pH-sensitive step of the enterovirus D68 lifecycle.

Enterovirus D68 (EV-D68) contributes significantly to pathogen-induced respiratory illnesses and severe neurological disorders like acute flaccid myelitis. We lack EV-D68 preventive measures, and knowledge of its molecular and cellular biology is incomplete. Multiple studies have highlighted the role of membrane compartments and autophagy during picornavirus multiplication. Galitska et al. found that EV-D68 also exploits cellular autophagic compartments and relies on autophagic machinery as pro-viral factors (G. Galitska, A. Jassey, M. A. Wagner, N. Pollack, et al., mBio e02141-23, 2023, https://doi.org/10.1128/mbio.02141-23). Surprisingly, failure of the autophagic compartment to acidify early during EV-D68 infection causes a delay in RNA synthesis that has not been reported for other enteroviruses. This delay appears to reflect the inability of viral proteins 2B and 3A to engage membranes stably, leading to their degradation in the cytoplasm. Observations like this underscore the importance of studying individual members of the virus genus. It will be interesting to understand how this phenomenon connects to EV-D68 pathogenesis, if at all.

Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

Maintenance of a host-specific minority mutation in the West Nile virus NS3.

West Nile virus (WNV), the most prevalent arthropod-borne virus (arbovirus) in the United States, is maintained in a cycle between Culex spp. mosquitoes and birds. Arboviruses exist within hosts and vectors as a diverse set of closely related genotypes. In theory, this genetic diversity can facilitate adaptation to distinct environments during host cycling, yet host-specific fitness of minority genotypes has not been assessed. Utilizing WNV deep-sequencing data, we previously identified a naturally occurring, mosquito-biased substitution, NS3 P319L. Using both cell culture and experimental infection in natural hosts, we demonstrated that this substitution confers attenuation in vertebrate hosts and increased transmissibility by mosquitoes. Biochemical assays demonstrated temperature-sensitive ATPase activity consistent with host-specific phenotypes. Together these data confirm the maintenance of host-specific minority variants in arbovirus mutant swarms, suggest a unique role for NS3 in viral fitness, and demonstrate that intrahost sequence data can inform mechanisms of host-specific adaptation.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

In Science Journals.

Highlights from the Science family of journals.

The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP.

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and ß phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems.

CRISPR/Cas-mediated genome editing in human pluripotent stem cells (hPSCs) offers unprecedented opportunities for developing in vitro disease modeling, drug screening and cell-based therapies. To efficiently deliver the CRISPR components, here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively. These vectors utilized the PiggyBac transposon system, which allows stable expression of CRISPR components in hPSCs. The Cas9 vector PB-CRISPR exhibited high efficiency (up to 99%) of inducing gene knockout in both protein-coding genes and long non-coding RNAs. The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown (98% knockdown for CD90) with optimized gRNA designs. Taken together, our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.

The Stem-Loop I of Senecavirus A IRES Is Essential for Cap-Independent Translation Activity and Virus Recovery.

Senecavirus A (SVA) is a picornavirus that causes vesicular disease in swine and the only member of the Senecavirus genus. Like in all members of Picornaviridae, the 5' untranslated region (5'UTR) of SVA contains an internal ribosome entry site (IRES) that initiates cap-independent translation. For example, the replacement of the IRES of foot-and-mouth disease virus (FMDV) with its relative bovine rhinitis B virus (BRBV) affects the viral translation efficiency and virulence. Structurally, the IRES from SVA resembles that of hepatitis C virus (HCV), a flavivirus. Given the roles of the IRES in cap-independent translation for picornaviruses, we sought to functionally characterize the IRES of this genus by studying chimeric viruses generated by exchanging the native SVA IRES with that of HCV either entirely or individual domains. First, the results showed that a chimeric SVA virus harboring the IRES from HCV, H-SVA, is viable and replicated normally in rodent-derived BHK-21 cells but displays replication defects in porcine-derived ST cells. In the generation of chimeric viruses in which domain-specific elements from SVA were replaced with those of HCV, we identified an essential role for the stem-loop I element for IRES activity and recombinant virus recovery. Furthermore, a series of stem-loop I mutants allowed us to functionally characterize discrete IRES regions and correlate impaired IRES activities, using reporter systems with our inability to recover recombinant viruses in two different cell types. Interestingly, mutant viruses harboring partially defective IRES were viable. However, no discernable replication differences were observed, relative to the wild-type virus, suggesting the cooperation of additional factors, such as intermolecular viral RNA interactions, act in concert in regulating IRES-dependent translation during infection. Altogether, we found that the stem-loop I of SVA is an essential element for IRES-dependent translation activity and viral replication.

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective.

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a 'polymerase' protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Single-cell analysis for the study of viral inhibitors.

Stochastic outcomes of viral infections are attributed in large part to multiple layers of intrinsic and extrinsic heterogeneity that exist within a population of cells and viruses. Traditional methods in virology often lack the ability to demonstrate cell-to-cell variability in response to the invasion of viruses, and to decipher the sources of heterogeneities that are reflected in the variable infection dynamics. To overcome this challenge, the field of single-cell virology emerged less than a decade ago, enabling researchers to reveal the behavior of single, isolated, infected cells that has been masked in population-based assays. The use of microfluidics in single-cell virology, in particular, has resulted in the development of high-throughput devices that are capable of capturing, isolating, and monitoring single infected cells over the duration of an infection. Results from the studies of viral infection dynamics presented in this chapter indicate how single-cell data provide a more accurate prediction of the start time, replication rate, duration, and yield of infection when compared to population-based data. Additionally, single-cell analysis reveals striking differences between genetically distinct viruses that are almost indistinguishable in population methods. Importantly, both the efficacy and distinct mechanisms of action of antiviral compounds can be elucidated by using single-cell analysis.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.