Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Tools for Rapid Genetic Engineering of Vibrio fischeri.

Vibrio fischeri is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for V. fischeri strain ES114, we anticipate that these tools can be adapted for use in other V. fischeri strains and, potentially, other microbes.IMPORTANCEVibrio fischeri is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension and then introduced directly into V. fischeri, eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in V. fischeri and potentially other Vibrio species.

Peptidomimetic Oligomers Targeting Membrane Phosphatidylserine Exhibit Broad Antiviral Activity.

The development of durable new antiviral therapies is challenging, as viruses can evolve rapidly to establish resistance and attenuate therapeutic efficacy. New compounds that selectively target conserved viral features are attractive therapeutic candidates, particularly for combating newly emergent viral threats. The innate immune system features a sustained capability to combat pathogens through production of antimicrobial peptides (AMPs); however, these AMPs have shortcomings that can preclude clinical use. The essential functional features of AMPs have been recapitulated by peptidomimetic oligomers, yielding effective antibacterial and antifungal agents. Here, we show that a family of AMP mimetics, called peptoids, exhibit direct antiviral activity against an array of enveloped viruses, including the key human pathogens Zika, Rift Valley fever, and chikungunya viruses. These data suggest that the activities of peptoids include engagement and disruption of viral membrane constituents. To investigate how these peptoids target lipid membranes, we used liposome leakage assays to measure membrane disruption. We found that liposomes containing phosphatidylserine (PS) were markedly sensitive to peptoid treatment; in contrast, liposomes formed exclusively with phosphatidylcholine (PC) showed no sensitivity. In addition, chikungunya virus containing elevated envelope PS was more susceptible to peptoid-mediated inactivation. These results indicate that peptoids mimicking the physicochemical characteristics of AMPs act through a membrane-specific mechanism, most likely through preferential interactions with PS. We provide the first evidence for the engagement of distinct viral envelope lipid constituents, establishing an avenue for specificity that may enable the development of a new family of therapeutics capable of averting the rapid development of resistance.

Persistent Coxsackievirus B3 Infection in Pancreatic Ductal Cells In Vitro Downregulates Cellular Polyamine Metabolism.

Picornaviruses infect a wide variety of cell types in vitro, with rapid replication kinetics and pronounced cytopathic effect. Coxsackievirus B3 (CVB3) can also establish a persistent infection in vivo that can lead to pathology, including dilated cardiomyopathy and myocarditis. One model system to study persistent infection is the pancreatic ductal cell line PANC-1, which CVB3 infects and is maintained indefinitely. We have characterized this model for CVB3 infection to study persistent infection for over 6 months. We find that CVB3 rapidly replicates within PANC-1 cells without robust cytopathic effect, and after 1 month in culture, titers stabilize. We find that infection does not significantly affect cellular viability. Persistent virus reverts to lytic infection when transferred to Huh7 or Vero cells. We find that persistent CVB3 adapts to PANC-1 cells via mutation of its capsid proteins and, curiously, the viral polymerase (3Dpol) to generate a high-fidelity polymerase. Persistent infection is associated with reduced cleavage of eIF4G, reduced plaque size, and decreasing particle infectivity. We further find that polyamine metabolism is altered in persistently infected cells, with the rate-limiting enzyme ornithine decarboxylase (ODC1) reduced in translation. We further find that targeting polyamine synthesis reduces persistent infection without affecting the viability of the PANC-1 cells. Finally, we find that viral fidelity is essential to maintaining CVB3 infection, and targeting viral fidelity reduces persistent virus infection. Together, these data highlight a novel role for polyamines and fidelity in persistent CVB3 infection and suggest avenues for therapeutic development to target persistent infection. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease, including cardiomyopathies. Viruses like coxsackievirus B3 (CVB3) can cause tissue damage by lytically infecting cells; however, CVB3 can also persistently infect, which has been associated with several pathologies. Studying persistent infection in vitro is challenging, as CVB3 lytically infects most cellular model systems. Here, we show that CVB3 establishes persistent infection in pancreatic ductal cells in vitro, similar to prior studies on other coxsackieviruses. We also show that this infection results in adaptation of the virus to these cells, as well as changes to cellular metabolism of polyamines.

A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency.

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2-the last two clustering with the eukaryotic eIF2α kinases-as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.

Polyamine-Linked Cholesterol Incorporation in Rift Valley Fever Virus Particles Promotes Infectivity.

Viruses rely on an array of cellular metabolites to replicate and form progeny virions. One set of these molecules, polyamines, are small aliphatic molecules, which are abundant in most cells, that support virus infection; however, the precise roles of polyamines in virus infection remain incompletely understood. Recent work demonstrated that polyamine metabolism supports cellular cholesterol synthesis through translation of the key transcription factor SREBP2. Here, we show that the bunyavirus Rift Valley fever virus (RVFV) relies on both cholesterol and polyamines for virus infection. Depletion of cellular cholesterol or interruption of cholesterol trafficking negatively impacts RVFV infection. Cholesterol is incorporated into RVFV virions and mediates their infectivity in a polyamine-dependent manner; we find that the virus derived from polyamine-depleted cells lacks cholesterol within the virion membrane. Conversely, we find that virion-associated cholesterol is linked to the incorporation of spermidine within the virion. Our prior work demonstrated that polyamines facilitate pH-mediated fusion and genome release, which may be a consequence of cholesterol depletion within virions. Thus, our work highlights the metabolic connection between polyamines and cholesterol synthesis to impact bunyavirus infection. These data demonstrate the connectedness between cellular metabolic pathways and reveal potential avenues of therapeutic intervention.

Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and Entry.

Coronaviruses first garnered widespread attention in 2002 when the severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from bats in China and rapidly spread in human populations. Since then, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged and still actively infects humans. The recent SARS-CoV-2 outbreak and the resulting disease (coronavirus disease 2019, COVID19) have rapidly and catastrophically spread and highlighted significant limitations to our ability to control and treat infection. Thus, a basic understanding of entry and replication mechanisms of coronaviruses is necessary to rationally evaluate potential antivirals. Here, we show that polyamines, small metabolites synthesized in human cells, facilitate coronavirus replication and the depletion of polyamines with FDA-approved molecules significantly reduces coronavirus replication. We find that diverse coronaviruses, including endemic and epidemic coronaviruses, exhibit reduced attachment and entry into polyamine-depleted cells. We further demonstrate that several molecules targeting the polyamine biosynthetic pathway are antiviral in vitro. In sum, our data suggest that polyamines are critical to coronavirus replication and represent a highly promising drug target in the current and any future coronavirus outbreaks.

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).

Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote Entry.

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules are diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively charged molecules involved in cell cycle, translation, and nucleic acid metabolism, among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related molecules associate with purified virions and transmit from cell to cell during infection. Virion-associated spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temperature sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions containing spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro.

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

Ribavirin Induces Polyamine Depletion via Nucleotide Depletion to Limit Virus Replication.

Common antivirals include nucleoside or nucleotide analogs with base prodrugs. The antiviral ribavirin, a US Food and Drug Administration (FDA)-approved nucleoside antimetabolite, halts guanine production, mutagenizes viral genomes, and activates interferon signaling. Here, we find that ribavirin induces spermidine-spermine N1-acetyltransferase (SAT1), a polyamine catabolic enzyme. Polyamines are small, positively charged molecules involved in cellular functions such as transcription and translation. Previous work showed that SAT1 activation and polyamine depletion interfere with RNA virus replication. We show ribavirin depletes polyamines via SAT1, in conjunction with its known mechanisms. SAT1 transcripts, protein, and activity are induced in a dose-dependent manner, which depletes polyamine levels and reduces viral titers. Inhibition of SAT1 activity, pharmacologically or genetically, reduces ribavirin's effectiveness against three virus infection models. Additionally, ribavirin-mediated polyamine depletion results from nucleotide pool depletion. These data demonstrate another mechanism of ribavirin that inform its clinical effectiveness, which may provide insight for improved therapies.