A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315.

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

HIV-1 Frameshift RNA-Targeted Triazoles Inhibit Propagation of Replication-Competent and Multi-Drug-Resistant HIV in Human Cells.

The HIV-1 frameshift-stimulating (FSS) RNA, a regulatory RNA of critical importance in the virus' life cycle, has been posited as a novel target for anti-HIV drug development. We report the synthesis and evaluation of triazole-containing compounds able to bind the FSS with high affinity and selectivity. Readily accessible synthetically, these compounds are less toxic than previously reported olefin congeners. We show for the first time that FSS-targeting compounds have antiviral activity against replication-competent HIV in human cells, including a highly cytopathic, multidrug-resistant strain. These results support the viability of the HIV-1 FSS RNA as a therapeutic target and more generally highlight opportunities for synthetic molecule-mediated interference with protein recoding in a wide range of organisms.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.