The APOBEC family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes – APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new host-pathogen conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.

Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or "shock" drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection. The suggested "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

Ebola virus (EBOV) is among the most devastating pathogens causing fatal hemorrhagic fever in humans. The epidemics from 2013 to 2016 resulted in more than 11,000 deaths, and another outbreak is currently ongoing. Since there is no FDA-approved drug so far to fight EBOV infection, there is an urgent need to focus on drug discovery. Considering the tight correlation between the high EBOV virulence and its ability to suppress the type I interferon (IFN-I) system, identifying molecules targeting viral protein VP24, one of the main virulence determinants blocking the IFN response, is a promising novel anti-EBOV therapy approach. Hence, in the effort to find novel EBOV inhibitors, a screening of a small set of flavonoids was performed; it showed that quercetin and wogonin can suppress the VP24 effect on IFN-I signaling inhibition. The mechanism of action of the most active compound, quercetin, showing a half-maximal inhibitory concentration (IC50) of 7.4 μM, was characterized to significantly restore the IFN-I signaling cascade, blocked by VP24, by directly interfering with the VP24 binding to karyopherin-α and thus restoring P-STAT1 nuclear transport and IFN gene transcription. Quercetin significantly blocked viral infection, specifically targeting EBOV VP24 anti-IFN-I function. Overall, quercetin is the first identified inhibitor of the EBOV VP24 anti-IFN function, representing a molecule interacting with a viral binding site that is very promising for further drug development aiming to block EBOV infection at the early steps.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome whose recent epidemic enlightened the urgent need of novel therapeutic agents, since no drug is currently approved. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which the viral protein VP35 plays a crucial role, blocking the interferon type I production, firstly by masking the viral dsRNA and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of VP35 interaction with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification, and a novel and robust fluorescence-based rVP35-RNA interaction assay (Z'-factor of 0.69). Taking advantage of such newlyestablished methods, we screened a small library of Sardinian natural extracts finding Limonium morisianum as the most potent inhibitor extract. A bio-guided fractionation led to the identification of myricetin as the component able to inhibit rVP35-dsRNA interaction with an IC 50 value of 2.7 µM.Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent VP35-dsRNA inhibitors.