Antiviral Research 171 (2019) 104613 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Review Article Ribonuclease H, an unexploited target for antiviral intervention against HIV and hepatitis B virus T ∗∗ ∗ Enzo Tramontanoa, , Angela Coronaa, Luis Menéndez-Ariasb, a Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy b Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain ARTICLE INFO ABSTRACT Keywords: Ribonucleases H (RNases H) are endonucleolytic enzymes, evolutionarily related to retroviral integrases, DNA Ribonuclease H transposases, resolvases and numerous nucleases. RNases H cleave RNA in RNA/DNA hybrids and their activity Reverse transcriptase plays an important role in the replication of prokaryotic and eukaryotic genomes, as well as in the replication of HIV reverse-transcribing viruses. During reverse transcription, the RNase H activity of human immunodeficiency Hepatitis B virus virus (HIV) and hepatitis B virus (HBV) degrades the viral genomic RNA to facilitate the synthesis of viral Antiviral double-stranded DNA. HIV and HBV reverse transcriptases contain DNA polymerase and RNase H domains that act in a coordinated manner to produce double-stranded viral DNA. Although RNase H inhibitors have not been developed into licensed drugs, recent progress has led to the identification of a number of small molecules with inhibitory activity at low micromolar or even nanomolar concentrations. These compounds can be classified into metal-chelating active site inhibitors and allosteric inhibitors. Among them, α-hydroxytropolones, N-hydro- xyisoquinolinediones and N-hydroxypyridinediones represent chemotypes active against both HIV and HBV RNases H. In this review we summarize recent developments in the field including the identification of novel RNase H inhibitors, compounds with dual inhibitory activity, broad specificity and efforts to decrease their toxicity. 1. Introduction integrase (IN), and (iii) the proteolytic activity of HIV protease (Menéndez-Arias, 2013). These drugs block viral replication, integra- Reverse-transcribing viruses include important human pathogens, tion and maturation, respectively. In addition, drugs targeting viral such as the human immunodeficiency viruses type 1 and type 2 (HIV-1 entry (e.g. coreceptor antagonists or fusion inhibitors) are used in sal- and HIV-2, respectively) and the hepatitis B virus (Menéndez-Arias vage therapies. Despite the success of antiretroviral therapy, acquired et al., 2017). HIV is the etiological agent of the acquired im- and transmitted drug resistance as well as long-term toxicity pose a munodeficiency syndrome (AIDS). The number of people living with major limitation to the long-term efficacy of current therapies, and HIV worldwide is very high with current estimates ranging between research towards exploiting other targets of antiviral intervention is still 32.7 and 44 million people (average 37.9 million in 2018). Although important for preparedness against future outcomes of the disease. the number of new infections has been steadily declining since the mid- Hepatitis B virus (HBV) infection remains an important cause of 90s, UNAIDS estimates that 1.7 million people got infected in 2018, morbidity and mortality. According to current estimates, there are more while the disease was globally responsible for around 770,000 deaths than 250 million people chronically infected with the virus (www.unaids.org). At present, no vaccine or cure is available, although (MacLachlan and Cowie, 2015; Seto et al., 2018) and twenty to thirty highly active antiretroviral therapy (HAART) has been highly effective percent of the chronically infected adults develop cirrhosis and liver in controlling viral load and preventing the onset of symptoms and cancer (Seto et al., 2018). HBV and associated liver diseases are re- progression to AIDS. sponsible for nearly 900,000 deaths each year, mostly from complica- Most of the currently approved anti-HIV drugs are inhibitors of tions (including cirrhosis and hepatocellular carcinoma) (WHO, 2017). enzymes encoded within the viral genome: (i) DNA polymerase activity Infection and development of chronic disease can be prevented by HBV of HIV reverse transcriptase (RT), (ii) strand transfer activity of HIV vaccination, which is most effective if given within 24 h after birth ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (E. Tramontano), [email protected] (L. Menéndez-Arias). https://doi.org/10.1016/j.antiviral.2019.104613 Received 29 August 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 Available online 21 September 2019 0166-3542/ © 2019 Elsevier B.V. All rights reserved. E. Tramontano, et al. Antiviral Research 171 (2019) 104613 (followed by two to three doses at monthly intervals). However, timely synthesis with the removal of the tRNA primer, also catalyzed by the infant vaccination has not been implemented everywhere and despite RNase H. progress, universal coverage is very difficult to achieve (Chang and Reverse transcription shows remarkable differences in HBV com- Nguyen, 2017). On the other hand, available treatments (long-term pared to HIV and other retroviruses (Fig. 1)(Hu and Seeger, 2015; nucleos(t)ide-analogue therapies) are safe and well tolerated, achieve Menéndez-Arias et al., 2017). However, in both viruses the RNase H potent viral suppression, and reduce the incidence of liver-related activity of the viral polymerase plays a prominent role. In HBV reverse complications (Menéndez-Arias et al., 2014; Levrero et al., 2018). transcription, the pregenomic RNA (pgRNA) is extensively degraded However, approved treatments act exclusively on viral replication by during minus-strand DNA synthesis, although the last cleavage occurs inhibiting DNA synthesis catalyzed by the HBV polymerase, and current away from the capped 5′ end, leaving an RNA sequence (direct repeat 1, research focuses on alternative targets for the development of anti-HBV or DR1) that is used as primer for plus-strand DNA synthesis (Nassal, drugs (Pei et al., 2017). 2008; Hu and Seeger, 2015). The RNA primer can also translocate to the Approximately 10% of the HIV-infected population worldwide is DR2 region at the 5’ end of the minus-strand DNA, thereby facilitating coinfected with HBV, although this figure can be higher in several re- the circularization of the DNA genome after the corresponding template gions of Southeast Asia (Singh et al., 2017). HIV reverse transcriptase switch (rcDNA formation). The conversion of rcDNA into covalently and HBV polymerase inhibitors constitute the backbone of antiviral closed circular DNA (cccDNA) involves a series of biochemical events therapies against both pathogens. These drugs block viral DNA synth- including the removal of the 18-nt long RNA primer used in plus- esis during reverse transcription, although an obligatory step in the stranded DNA synthesis, but the molecular mechanisms have not been process involves the degradation of viral RNA by the associated ribo- elucidated and the participation of the viral RNase H is not clear yet nuclease H (RNase H) activity of the HIV and HBV polymerases. To (Hu and Seeger, 2015; Menéndez-Arias et al., 2017). date, RNase H remains as an unexploited target in antiviral interven- tion, and despite efforts to identify specific inhibitors, none of those 3. RNase H function and structure compounds has been approved for clinical use. In this review, we will summarize current knowledge on the structure and catalytic me- RNases H are among the most abundant proteins on our planet. chanism of RNases H, and efforts to discover and develop compounds RNases H bind to RNA/DNA hybrids in a sequence-nonspecific manner targeting the RNase H activity of HIV and HBV. and degrade the RNA strand. Eukaryotic RNase H was initially dis- covered in calf thymus lysates, although its role was unknown for a long 2. RNase H and reverse transcription time (Stein and Hausen, 1969). Its involvement in DNA replication was later suggested after being found in Saccharomyces cerevisiae (Karwan RNases H constitute a family of non-sequence-specific endonuclease and Wintersberger, 1986). enzymes that catalyze the cleavage of RNA in RNA/DNA substrates According to their structural features and evolutionary relation- through a hydrolytic mechanism. Members of the RNase H family can ships, RNases H have been classified into two major groups (RNase H1 be found in nearly all organisms, from bacteria to archaea to eukaryotes or HI and RNase H2 or HII). Arabic numerals have been traditionally (Majorek et al., 2014; Moelling et al., 2017). These enzymes are in- used to designate eukaryotic RNases H (H1 and H2), while Roman volved in many processes such as transposition, replication and repair numbers were assigned to prokaryotic enzymes (RNase HI and HII). of DNA, homologous recombination and RNA-mediated gene silencing. Type 1 RNases H are found in eukaryotic, bacterial and viral proteins, Soon after the discovery of retroviral RTs, its associated RNase H including those associated with RTs (e.g. HIV RT and HBV polymerase). activity was identified as an essential component of the enzyme Type 2 RNases are also found in eukaryotes, as well as in prokaryotes, (Mölling et al., 1971). Retroviral RTs contain DNA polymerase and including archaea. A third related class (designated RNase HIII) and RNase