Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox.

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 µM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

Two-Center Evaluation of Disinfectant Efficacy against Ebola Virus in Clinical and Laboratory Matrices.

Ebola virus (EBOV) in body fluids poses risk for virus transmission. However, there are limited experimental data for such matrices on the disinfectant efficacy against EBOV. We evaluated the effectiveness of disinfectants against EBOV in blood on surfaces. Only 5% peracetic acid consistently reduced EBOV titers in dried blood to the assay limit of quantification.

Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection.

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

The master regulator of the cellular stress response (HSF1) is critical for orthopoxvirus infection.

The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Closing the genome of Teredinibacter turnerae T7902 by long-read nanopore sequencing.

We present the complete closed circular genome sequence derived from Oxford Nanopore sequencing of the shipworm endosymbiont Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of host-symbiont evolution.

Outer membrane vesicles can contribute to cellulose degradation in Teredinibacter turnerae, a cultivable intracellular endosymbiont of shipworms.

Teredinibacter turnerae is a cultivable cellulolytic Gammaproeteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose, and pectin and contribute to lignocellulose digestion in the shipworm gut. However, the mechanism by which symbiont-made enzymes are secreted by T. turnerae and subsequently transported to the site of lignocellulose digestion in the shipworm gut is incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce outer membrane vesicles (OMVs) that contain a variety of proteins identified by LC-MS/MS as carbohydrate-active enzymes with predicted activities against cellulose, hemicellulose, and pectin. Reducing sugar assays and zymography confirm that these OMVs retain cellulolytic activity, as evidenced by hydrolysis of CMC. Additionally, these OMVs were enriched with TonB-dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations suggest potential roles for OMVs in lignocellulose utilization by T. turnerae in the free-living state, in enzyme transport and host interaction during symbiotic association, and in commercial applications such as lignocellulosic biomass conversion.

Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences.

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73-77, 2001; Colebunders and Borchert, J. Infect. 40:16-20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148-S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643-1645, 2005; Schornberg et al., J. Virol. 80:4174-4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163-175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae.

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.

The biosecurity benefits of genetic engineering attribution.

Biology can be misused, and the risk of this causing widespread harm increases in step with the rapid march of technological progress. A key security challenge involves attribution: determining, in the wake of a human-caused biological event, who was responsible. Recent scientific developments have demonstrated a capability for detecting whether an organism involved in such an event has been genetically modified and, if modified, to infer from its genetic sequence its likely lab of origin. We believe this technique could be developed into powerful forensic tools to aid the attribution of outbreaks caused by genetically engineered pathogens, and thus protect against the potential misuse of synthetic biology.

A Semi-automated High-Throughput Microtitration Assay for Filoviruses.

The 50% tissue culture infectious dose (TCID50) endpoint dilution assay is one of the gold standard methods for measuring filovirus infectivity. We have increased virology microtitration assay throughput at biosafety level (BSL)-4 by implementing automated liquid handling and semi-automated assay endpoint readout. Utilization of automated liquid handling for cell plating and virus dilution along with optimization of the assay endpoint readout, using a luminescent-based cell viability assay and an automated plate reader, has improved workflow efficiency, reduced operator burden and assay time, decreased assay variability, and increased data return.

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication.

UNLABELLED: Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates : EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein. IMPORTANCE: Ebolavirus (EBOV) is one of the most lethal human pathogens known. EBOV requires host factors for replication due to its small RNA genome. Here we show that the host protein eIF5A in its activated form is necessary for EBOV replication. We further show that the mechanism is through the accumulation of a single EBOV protein, VP30. To date, no other host proteins have been shown to interfere with the translation or stability of an EBOV protein. Activated eIF5A is the only protein in the cell known to contain the specific modification of hypusine; therefore, this pathway is a target for drug development. Further investigation into the mechanism of eIF5A interaction with VP30 could provide insight into therapeutics to combat EBOV.

Differences in the Comparative Stability of Ebola Virus Makona-C05 and Yambuku-Mayinga in Blood.

In support of the response to the 2013-2016 Ebola virus disease (EVD) outbreak in Western Africa, we investigated the persistence of Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 (EBOV/Mak-C05) on non-porous surfaces that are representative of hospitals, airplanes, and personal protective equipment. We performed persistence studies in three clinically-relevant human fluid matrices (blood, simulated vomit, and feces), and at environments representative of in-flight airline passenger cabins, environmentally-controlled hospital rooms, and open-air Ebola treatment centers in Western Africa. We also compared the surface stability of EBOV/Mak-C05 to that of the prototype Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (EBOV/Yam-May), in a subset of these conditions. We show that on inert, non-porous surfaces, EBOV decay rates are matrix- and environment-dependent. Among the clinically-relevant matrices tested, EBOV persisted longest in dried human blood, had limited viability in dried simulated vomit, and did not persist in feces. EBOV/Mak-C05 and EBOV/Yam-May decay rates in dried matrices were not significantly different. However, during the drying process in human blood, EBOV/Yam-May showed significantly greater loss in viability than EBOV/Mak-C05 under environmental conditions relevant to the outbreak region, and to a lesser extent in conditions relevant to an environmentally-controlled hospital room. This factor may contribute to increased communicability of EBOV/Mak-C05 when surfaces contaminated with dried human blood are the vector and may partially explain the magnitude of the most recent outbreak, compared to prior outbreaks. These EBOV persistence data will improve public health efforts by informing risk assessments, structure remediation decisions, and response procedures for future EVD outbreaks.

Probing the virus host interaction in high containment: an approach using pooled short hairpin RNA.

The study of viruses in high containment offers unique challenges for technology-intense approaches. These approaches include high-throughput screening for small-molecule antivirals and genetic perturbation-based screens for host factors required for viral replication. Here, we describe the use of whole-genome scale pooled short hairpin RNA (shRNA) libraries to screen for host factors necessary for viral infection at BSL2, and the transition of this technique into the BSL4 environment. Pooled screening provides a unique way to circumvent many of the technological challenges associated with other high-throughput screening approaches in high containment. Our pooled screening approach identified host factors involved in the replication of orthopoxviruses (Vaccinia and Monkeypox) and filoviruses (Ebola and Marburg) under conditions that enable straightforward screen-to-follow-up approaches.

Vaccinia reporter viruses for quantifying viral function at all stages of gene expression.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

Activation of stress response pathways promotes formation of antiviral granules and restricts virus replication.

The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and nuclear HNRNPs) and the response to cellular stress (stress granules and ND10 bodies). To better understand the role of stress-induced granules in viral infection, we have studied the ability of cells to restrict poxvirus replication through the formation of antiviral granules (AVGs). Of cells infected with a wild-type poxvirus, a small number spontaneously formed AVGs. In these AVG-positive cells, viral gene expression was inhibited. The addition of compounds that altered RNA helicase activity, induced oxidative stress, or stimulated translation initiation factor phosphorylation significantly increased the number of AVG-positive cells. When AVGs formed, both viral translation and titers were decreased even when host translation persisted. Treatment with the antiviral compound isatin ß-thiosemicarbazone (IBT), a compound that was used to treat smallpox infections, induced AVGs, suggesting a role for these structures in the pharmacological inhibition of poxvirus replication. These findings provide evidence that AVGs are an innate host response that can be exogenously stimulated to combat virus infection. Since small molecules are able to stimulate AVG formation, it is a potential target for new antiviral development.

Identification of a broad-spectrum inhibitor of viral RNA synthesis: validation of a prototype virus-based approach.

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify chemical scaffolds for the development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses, suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling.

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.