Understanding Helicases As a Means of Virus Control
Total Page：16
File Type：pdf, Size：1020Kb
Current Pharmaceutical Design, 2006, 12, 1315-1338 1315 Understanding Helicases as a Means of Virus Control D. N. Frick* and A. M. I. Lam Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, NY 10595, USA Abstract: Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, se- vere acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by prevent- ing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a re- quired cofactor. Key Words: ATPase, DExD/H-box proteins, motor proteins, enzyme mechanism. INTRODUCTION translation, disruption of RNA-protein complexes, and pack- aging of nucleic acids into virions. Most importantly, their Viruses evolve rapidly to evade not only the human im- validity as antiviral drug targets was recently confirmed mune system but also antiviral drugs. Although it is some- when compounds that inhibit a helicase encoded by herpes times possible to use only a single antiviral drug to control simplex virus (HSV) were shown to block viral replication DNA viruses that proofread and repair their genomes, single and disease progression in animal models [1, 2]. agents are rarely effective for viruses that evolve more rap- idly. The most successful method to control such a virus is to Most of the best-studied viral helicases are encoded by simultaneously target multiple loci with several agents. Such large DNA viruses or bacteriophage. In fact, some of the first combination therapies successfully control human immu- helicases identified over two decades ago were purified from nodeficiency virus (HIV) replication to delay the develop- these organisms [3-5]. Similar proteins are encoded by sev- ment of AIDS, and are presently the standard of care for HIV eral important human pathogens like HSV and human pa- patients. Similar approaches could be used to combat other pillomavirus (HPV). About 10 years after the discovery of viruses, but the technique is limited because most present DNA helicases, similar proteins were found to be made by antiviral drugs function by inhibiting either viral proteases, RNA viruses [6]. Most species of positive sense single- which are needed to process viral proteins, or nucleic acid stranded RNA ((+)ssRNA) viruses encode a helicase, and polymerases, which are required to copy the viral genome. several of these proteins have now been extensively studied. Although a few other antiviral drugs function by blocking The most detailed structural and mechanistic studies have viral entry or by modulating the host immune system, more been performed using the RNA helicase encoded by hepatitis drug targets are clearly needed. This review focuses on the C virus (HCV). A few other helicases from pathogenic hu- development of another critical protein synthesized by many man RNA viruses have also been studied in detail including viruses, an enzyme called helicase, as a potential antiviral those from severe acute respiratory syndrome coronavirus drug target. (SARS-CoV), Dengue fever virus (DFV), Japanese en- cephalitis virus (JEV), and West Nile virus (WNV). Curi- Helicases are motor proteins that use energy derived ously, no retroviruses or negative sense single-stranded RNA from ATP hydrolysis to separate nucleic acid strands. Such ((-)ssRNA) viruses have been reported to encode the synthe- an activity is necessary at several points during genome rep- sis of a helicase. Since the members of these classes replicate lication. In viruses with duplex nucleic acid genomes, the in the nucleus, they might simply utilize helicases encoded double helix must be separated for copying. Conversely, by the host instead of their own proteins. Indeed, HIV repli- viruses with single-stranded genomes must separate duplexes cation has recently been shown to require the human DDX3 that form after genome replication. Helicases have also been DEAD-box RNA helicase [7]. Large DNA viruses that in- shown to be required in transcription of viral mRNAs, vade the nucleus, like HSV, frequently bring with them a full complement of replication proteins, including at least one *Address correspondence to this author at the Department of Biochemistry helicase. Viruses that replicate outside the nucleus almost and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA; Tel: 914-594-4190; Fax: 914-594-4058; always encode a helicase, without which they cannot sur- E-mail: [email protected] vive. 1381-6128/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1316 Current Pharmaceutical Design, 2006, Vol. 12, No. 11 Frick and Lam Developing non-toxic helicase inhibitors is considerably This review will summarize the various viral helicases more difficult than developing drugs designed to inhibit that have been characterized to date, their evolutionary rela- other viral enzymes. Problems arise from several areas. First, tionships, mechanisms of action, and any inhibitors that have compared with proteases and polymerases, the mechanisms been reported in the scientific literature. The helicase litera- of the reactions catalyzed by helicases are still not as clearly ture is rapidly expanding but fortunately the field is fre- understood. Second, the helicase ATP-binding site is con- quently reviewed, and the reader will be directed to relevant served not only in all classes of helicases, but also in motor reviews in appropriate sections. It should also be noted at proteins, small GTPases, kinases, the AAA+ family (AT- this point that Delagoutte & von Hippel have recently re- Pases associated with various cellular activities), and even viewed the entire helicase field in an extensive two-part re- the mitochondrial ATP synthase (F1 ATPase) (Fig. (1)). view that is highly recommended [9, 10]. Thus, compounds that inhibit helicases via their ATP- binding sites could be quite toxic. Third, the role of helicases VIRAL HELICASE CLASSIFICATION in the viral lifecycle is still not well-defined. Although, in The best way to understand the bewildering amount of vitro all helicases are able to separate a nucleic acid strand from its complement, their movements could also rearrange viral helicase information is to realize that all helicases, from secondary structures or dislodge nucleic acid binding pro- both viruses and cellular organisms, share many common teins. Finally, the traditional assays measuring helicase- properties. Understanding these features will provide the catalyzed unwinding are tedious, making inhibitor screening basis for understanding the mechanism of action of these time-consuming. In the past few years, considerable progress complex enzymes. It should also be recognized that nature has been made in the area of assay development (see refer- has used the basic building blocks shared by all helicases to ence [8] for a review) and it is now possible to identify po- manipulate nucleic acids in many different ways and for tent helicase inhibitors using high throughput screening. The many different purposes. As a result, different helicase fami- challenge now is to understand how these compounds inter- lies have evolved that share little resemblance, at least super- act with helicases so that they can be developed into actual ficially. Thus, to really understand viral helicases, one must drugs. understand the common properties shared by all helicases, and the distinctive properties that characterize the various Fig. (1). Evolutionary relationship of viral helicases. All known viral helicases belong either to one of three helicase superfamilies or to the RecA/F1-ATPase superfamily. The five families of viral helicases from DNA viruses and three families of viral helicases from RNA viruses are highlighted with grey bars. Prototypes of each family are listed in parentheses. SF3 and DnaB-like helicases are fundamentally different from SF1 and SF2 helicases in that they contain only one RecA-like domain per subunit and must form rings and/or filaments to catalyze ATP hydrolysis. All helicases are in the ASCE subdivision of P-loop NTPases and share many basic features with the numerous other pro- tein families listed. The diagram is based on information found in references [234, 235]. Understanding Helicases as a Means of Virus Control Current Pharmaceutical Design, 2006, Vol. 12, No. 11 1317 helicase families. If a new viral pathogen is discovered, its discussed based on their functional similarities rather than genome sequence can be used to predict not only if the virus their