Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug ...targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. On the basis of this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.
The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription ...factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCFβ-TrCP, since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.
Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase and as an essential component required for processive DNA synthesis. The VACV A20 protein has no ...known catalytic function itself but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the inner core of the protein. Our mutational analysis revealed that a tripeptide motif (215GFI217) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly215 that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that 215GFI217 anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.
The 3′ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite (sat)C, terminate with the motif CCUGCCC-3′. Transcripts of satC with a deletion of the motif are repaired ...to wild type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3′end of the TCV genomic RNA. Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3′terminal CCC replaced by eight nonviral bases were repaired in plants by homologous recombination between the similar 3′ends of satC and TCV. Transcripts with deletions of four or five 3′terminal bases, in the presence or absence of nonviral bases, generated progeny with a mixture of wt and non-wt 3′ends in vivo. In vitro, RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3′end of truncated satC templates. The nontemplate additions at the 5′ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate, and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of three different repair mechanisms for TCV suggests that 3′end reconstruction is critical to virus survival.
Because oral transmission of SARS-CoV-2 is 3–5 orders of magnitude higher than nasal transmission, we investigated debulking of oral viruses using viral trap proteins (CTB-ACE2, FRIL) expressed in ...plant cells, delivered through the chewing gum. In omicron nasopharyngeal (NP) samples, the microbubble count (based on N-antigen) was significantly reduced by 20 μg of FRIL (p < 0.0001) and 0.925 μg of CTB-ACE2 (p = 0.0001). Among 20 delta or omicron NP samples, 17 had virus load reduced below the detection level of spike protein in the RAPID assay, after incubation with the CTB-ACE2 gum powder. A dose-dependent 50% plaque reduction with 50–100 ng FRIL or 600–800 μg FRIL gum against Influenza strains H1N1, H3N2, and Coronavirus HCoV-OC43 was observed with both purified FRIL, lablab bean powder or gum. In electron micrographs, large/densely packed clumps of overlapping influenza particles and FRIL protein were observed. Chewing simulator studies revealed that CTB-ACE2 release was time/dose-dependent and release was linear up to 20 min chewing. Phase I/II placebo-controlled, double-blinded clinical trial (IND 154897) is in progress to evaluate viral load in saliva before or after chewing CTB-ACE2/placebo gum. Collectively, this study advances the concept of chewing gum to deliver proteins to debulk oral viruses and decrease infection/transmission.
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Twitter headline: Chewing gum neutralizing both SARS-CoV-2 and influenza viruses is advanced to clinical testing.
We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and ...cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.
•The small molecule 7269 is the first antiviral specific for Molluscum contagiosum.•Exhaustive med chem failed to generate a second compound for drug development.•The impasse was solved by conjugating a Tri-Valine peptide to 7269.•The TriVal-7269 molecule has greater antiviral potency and is not toxic to cells.•Peptide-conjugated drugs represent a new approach to antiviral drug development.
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