Viral Polymerases: Structures, Functions and Roles as Antiviral Drug Targets

India

Preface

Satya P. Gupta, Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India

Viral polymerases constitute a vital component in the life cycle of many viruses, such as human immunodeficiency virus (HIV), hepatitis viruses, and influenza virus. They are essentially required for the replication of viruses. Polymerases are enzymes that synthesize long chains or polymers of nucleic acids. They are basically categorized as DNA or RNA polymerases and are used to assemble DNA and RNA molecules, respectively, by copying a DNA or RNA template strand using base–pairing interactions. Thus, the polymerases that can be found in viruses (called viral polymerases) represent favorable targets for the design and development of antiviral drugs. The book in hand presents chapters dealing with polymerases present in different viruses along with their structures, functions, and roles as antiviral drug targets. There are 15 chapters written by highly acclaimed authors. In Chapter 1, RNA-Dependent RNA Polymerases and Their Emerging Roles in Antiviral Therapy, Ganeshpurkar et al. present a detail of RNA-dependent RNA polymerases and how they could be exploited to develop different antiviral drugs. In Chapter 2, Structure–Function Relationship of Negative-Stranded Viral RNA Polymerases: Prospectives for Antiviral Therapy, Tomar et al. discuss particularly the specific role of negative-stranded RNA polymerases in the life cycle of some viruses and their roles in antiviral therapy. Negative-stranded RNA viruses, which may have segmented or nonsegmented genomes, are to be blamed for plentiful of grave viral diseases both in humans and animals, such as measles, rabies, influenza, Crimean Congo hemorrhagic fever, Ebola and Lassa fevers. In Chapter 3, RNA-Dependent RNA Polymerase of Alphaviruses: A Potential Target for the Design of Drugs Against Alphaviruses, Tomar et al. narrate the specificity of RNA-dependent RNA polymerase (RdRP) present in alphaviruses and presented a few identified inhibitor molecules and strategies that can be undertaken for the development of new molecules that can potentially inhibit alphavirus RdRP. Alphaviruses have gained popularity in a very short duration of time because of repeated epidemics in different regions of the world, but unfortunately there has been no effective chemotherapy against this family of viruses. Another class of polymerases, DNA-dependent DNA polymerases, are multifunctional enzymes with polymerase and exonuclease activities and constitute good targets to develop antiviral drugs. Therefore, in Chapter 4, DNA-Dependent DNA Polymerases as Drug Targets in Herpesviruses and Poxviruses, Luczkwiak et al. provide a general overview of the biochemical and structural properties of herpesvirus and poxvirus DNA polymerases and the development of potent antiviral drugs based on them. In Chapter 5, Polio Virus Polymerase: An Effective Target for Design and Development of Anti-Polio Drugs, Dutta Gupta and Banerjee describe structure and function of poliovirus RNA-dependent RNA polymerase with a future perspective in the discovery of novel drug molecules against polio. Studies on HIV-1 polymerase and its inhibitors have been beautifully presented by Gupta and Balasubramanian in Chapter 6, Studies on HIV-1 Polymerase and Its Inhibitors. HIV-1 RT is a multifunctional enzyme that contains DNA polymerase and RNase active sites. The chapter describes how both can be exploited to design potent anti-HIV-1 drugs.

The Ebolavirus causes an acute, serious illness which is often fatal if untreated. The 2014–2016 outbreak in West Africa was the largest and most complex Ebola outbreak since the first discovery of this virus in 1976. Therefore in Chapter 7, A Focus on Ebola Virus Polymerase: Structure, Functions and Antiviral Therapies, Pettini et al. present an overview about the Ebolavirus RNA polymerase, describing its structure, function, and inhibitors. Hepatitis C virus (HCV) infection is frequently associated with the development of chronic liver disease, liver cirrhosis, and hepatocellular carcinoma. Chapter 8, Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase: An Integral Part of HCV Antiviral Therapy, Dash et al., therefore, summarize recent progress made in understanding the structure and function of NS5B polymerase, an RNA-dependent RNA polymerase present in HCV, and the development of targeted NS5B inhibitors widely used in the treatment of chronic HCV infection. Another member of the family of hepatitis viruses, equally deadly, is hepatitis B virus (HBV). Bhise describes in Chapter 9, HBV Polymerase as a Target for Development of Anti-HBV Drugs, the structure and function of DNA polymerase present in HBV and its role as a target for development of anti-HBV drugs. Some medicinal plants exhibiting anti-HBV action have also been indicated in this chapter. Coronaviruses (CoVs) are a major group of viruses known to be responsible for wide spectrum of diseases in multiple species. They require RdRPs for various steps in their life cycle and thus Anand and Al-Nema in Chapter 10, Polymerases of Coronaviruses: Structure, Function, and Inhibitors, discuss in detail the structure and functions of CoV polymerases and the development of their potential inhibitors.

Human rhinovirus is responsible for causing 50% of common cold infections in infants and adults and its replication is greatly helped by an RNA-dependent RNA polymerase known as 3Dpol. Owing to the presence of over 100 serotypes of this enzyme, designing specific inhibitors targeting it is a challenging task, but Nirwan and Kakkar highlight its structure and functions as well as its potent inhibitors studied till date in Chapter 11, Rhinovirus RNA Polymerase: Structure, Function and Inhibitors. Herpes simplex viruses (HSV1 and HS2) are the second leading cause of human viral diseases. Therefore, in Chapter 12, Herpes Virus Polymerase Inhibitors, Das and Hong present the development of antiviral therapy based on the inhibition of DNA polymerase present in herpesviruses. The development of HSV therapies based on the inhibition of HSV polymerase is an important topic in medicinal chemistry research nowadays. Among the human infections transmitted by mosquitos, Zika virus (ZIKV) infection has potential as worldwide pandemic. Therefore, in Chapter 13, Zika Virus Polymerase: Structure, Function, and Inhibitors, Vaishali et al. discuss in detail the structural and functional aspects of an NS5 polymerase present in zika virus and the development of its inhibitors. In Chapter 14, Dengue Virus Polymerase: A Crucial Target for Anti-Viral Drug Discovery, Patil et al. also dwell upon the structure and function of an NS5 polymerase in dengue virus, a most prevalent mosquito-borne viral pathogen, and present the details of development of potent inhibitors of this polymerase to combat the dengue virus infection. In the last chapter, Chapter 15, Adenovirus DNA Polymerase: Structure, Function and Prospects in Diagnostics and Therapeutics, Kumar et al. present the details of DNA polymerase present in adenoviruses. Adenoviruses cause variety of infections in mammals and birds leading to high morbidity and mortality in immunocompromised hosts. They also point out that adenovirus plomerase (Ad pol) is a processive enzyme and in addition to a polymerase activity it also possesses 3ʹ-5ʹ exonuclease activity. In recent years, it has emerged as a promising target for the development of diagnostic and therapeutic agents against adenoviral diseases. Thus, the book covers a detailed description of structures and functions of specific polymerases present in different viruses and their roles in the development of antiviral chemotherapy. It is, therefore, hoped that the book will be highly useful to those working in the areas of biochemistry, virology, medicinal chemistry, enzymology, immunology, and pharmacology and particularly to those involved in the design and development of antiviral therapy. I have highly enjoyed reading all the articles contributed by learned contributors and hope so would all the readers do. I highly thank all the contributors and desire such nice cooperation from them in future, too.

Chapter 1

RNA-Dependent RNA Polymerases and Their Emerging Roles in Antiviral Therapy

Ankit Ganeshpurkar, Gopichand Gutti and Sushil Kumar Singh,    Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi, Uttar Pradesh, India

Abstract

Viruses are the causative agents leading to variety of diseases. The viral RdRPs are the attractive targets for antiviral therapy. This chapter presents a collective view on the structure and ligand-based drug design for the inhibition of viral RdRPs. It also highlights the structural aspects of various RdRPs. Interestingly, all these enzymes share structural similarities but certain significantly different structural features may be vital to design their selective inhibitors. The chapter also introduces the readers with different classes of RdRP inhibitors, i.e., nucleoside and nonnucleoside inhibitors involving various scaffolds such as Anthranilic acid, Benzimidazole, Benzothiadiazine Indole, Proline sulfonamide, Pyrrolidine diacids, and Pyridobenzothiazole. This chapter is thus prelude to the remaining chapters in this book, which deal with the viral RdRPs.

Keywords

RNA-dependent RNA polymerase; nucleoside inhibitors; allosteric inhibitors; structure-based drug design

1.1 Introduction

Viruses are pestilential agents, typically made up of one kind of nucleic acid, either RNA or DNA. They attach to the host cell to invade it and replicate inside the cell by utilizing host biochemicals and processes. Among the two types of viruses, RNA and DNA viruses, the former cause a critical burden on world healthcare systems (Cameron et al., 2009; Claverie, 2006). Viruses escape the host immune response by integrating their genome into the host DNA and due to this ability they play the crucial roles of causative agents for plentiful human diseases (Hemmi et al., 2002; Hancioglu et al., 2007; Haseltine et al., 2008; Jensen and Thomsen, 2012; Ahlquist et al., 2003). RNA viruses use well-organized replication and transcription strategies to amplify their genetic material (Díez et al., 2000). The evolutionary dynamics of RNA viruses complicate the management of viral diseases including hepatitis, dengue, yellow fever, Ebola, etc., and the mutation rate in these viruses is perilously equal to the highest tolerable error rate (Hiscox, 2007; Holmes, 2011). RNA viruses have a compact and well-organized genome sequences to maintain mutational robustness (Franco et al., 1995). The theory, Quasispecies, explains about the viral population, which is flexible with diverse group of variants and different replicative capacities will emerge with spontaneous mutations after each round of replication. The theory also has been used to describe the evolutionary dynamics of RNA viruses (Lauring and Andino, 2010; Lauring et al., 2013). A few examples of medically important RNA viruses include Zika virus (ZIKV), which spreads by daytime-active Aedes mosquitoes; Ebola virus, which causes deadly hemorrhagic fevers; influenza virus, which causes notorious pandemic Spanish flu infection; and hepatitis C virus, a blood-borne virus, which causes both acute and chronic infections. Globally, an estimated 71 million people have chronic hepatitis C infection. Although, several viral drug targets are known and well established, no single agent has been discovered for complete cure of viral infections. Several challenges still exist in the drug discovery cascade of the antiviral. Despite of continuous advances, RNA viruses remain major causes of human disease that costs in mortality, morbidity, and monetary terms (Neumann et al., 2002; Ahlquist, 2006; Cattaneo et al., 1988). The RNA viruses are classified into three different classes, i.e., double-stranded (dsRNA), positive sense single-strand (+ssRNA), and negative sense single-strand (–ssRNA) based on type of genome (Ahlquist, 2002; Baltimore, 1971).

1.2 RNA Polymerase

RNA, a polynucleotide molecule, is one of the major essential constituents of life. It is often present in single-stranded form and performs a variety of functions not limited to only peptide bond formation but can also be involved in replication, intron splicing, translational regulation, and gene regulation. RNAs are of three types, viz. messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). The mRNA conveys information stored in genetic material to direct protein synthesis, rRNA helps to form peptide linkage between amino acids while tRNA helps to supply amino acids to the ribosomes. An RNA molecule having conserved base pair regions, plays many important roles. The most stable helix may have important biological roles. RNA molecules replicate through a class of enzymes termed as RNA polymerases. The RNA polymerases belong to two different classes as described below.

1.2.1 DNA-Dependent RNA Polymerases

The RNA polymerases that catalyze the formation of phosphodiester bonds between ribonucleotides to form an ssRNA polymer using DNA as a template are called as DNA-dependent RNA polymerases (DdRPs). They are found in various life forms including viruses. They uncoil the double-stranded DNA, which acts as a template for the synthesis of RNA.

1.2.2 RNA-Dependent RNA Polymerases

The RNA-dependent RNA polymerases (RdRPs), also called RNA replicases, catalyze the replication of RNA from an RNA template and are essential proteins encoded in the genomes of all RNA-containing viruses with no DNA stage (Poltronieri et al., 2015; Ahlquist, 2006). An RdRP is involved in a pathway outside the central dogma of early molecular biology. RdRPs, present in a wide variety of RNA viruses, are involved in genome replication, mRNA synthesis, or RNA recombination, etc. They are essential for the survival of viruses. They also play an important role in evolution when RNA was primary genetic material. The RdRPs have multiple conserved sequences and motifs across the length. They share structural similarity with DNA-dependent DNA polymerase, DdRP, and reverse transcriptase (RT). The higher error rate (in order of 10−4) in transcription process by RdRPs leads to genomic variations in the RNA virus population. The switching mechanism during RdRPs copying process may lead to RNA recombination and also helps viruses to repair deleterious mutations and acquire new genes and genetic rearrangements.

1.3 Structure of Viral RNA-Dependent RNA Polymerases

All RdRPs share structural resemblance, having fingers, palm, and thumb domains, which provide cupped right-hand-like appearance. The three domains together constitute the binding site for RNA and nucleotide phosphates (NTPs). Although, the low sequence similarity is observed in various polymerases of different families, they have similar structural features and thus indicate a high evolutionary similarity in distinct species. A peculiar N-terminal sequence is found between fingers and thumb domain that appears as a bridge in all RdRPs. Cystoviridae, Flaviviridae, and Reoviridae family of viruses show the presence of C-terminal domain encircling the central cleft of the enzyme. The representative RdRP structure (EV71) has seven motifs or specific regions, i.e., G, F1–3, A, B, C, D, and E from N- to C-termini. Each motif has different sequences, but their folds are conserved and adopt specific conformation, which is similar in different RdRPs (homomorphs). Loops that connect the finger domains are called fingertips and are responsible for overall unique closed-hand conformation of the enzyme. They are not present in other types of polymerases. The fingertip residues pack the major groove of the template RNA and unstack the RNA strand at +3 position. These residues are not conserved but play an important role in holding the template RNA without much conformational changes. The variations in finger subdomain also help in performing functions like nuclear import, oligomerization, phosphorylation, and protein–protein interactions.

The thumb domain residues are involved in RNA binding and surround the minor groove of the template RNA while other residues also contribute to the formation of NTP tunnel. The entire domain has α-helices except the N-terminal that bears β-sheet only (Hansen et al., 1997). This region has small sequence protrusions that stabilize template channel and initiate binding of NTPs on the template. These regions have higher conformational flexibility, which facilitates translocation of the template after each NTP addition (condensation reaction). The NTP tunnels are lined by positively charged amino acid residues, though not sufficient, but still provide guidance to NTPs to enter the catalytic site (Fig. 1.1A).

Figure 1.1 (A) Structure of RdRP, representing the active site (EV71); (B) structural representation of major cavities of RdRP (EV71); (C) ribbon structure of EV71 RdRP depicting the various subdomains of RdRPs (PDB–3N6M); and (D) two metal ions mechanism for stabilization of incoming NTP.

The palm domain consists of A, C, and D motifs bearing catalytic site. The three β-antiparallel sheets lined by three α-helices form the core of RdRPs, which is conserved in all the enzymes. The core region also has highly conserved N-terminal aspartate residues, i.e., Dx4–5D and xDD sequences (x represents any amino acid residue) of motifs A and C. Further, deletion or alteration of these residues leads to a decrease or complete loss in the enzyme activity. For instance, the mutation of arginine to lysine results in decreased activity of hepatitis C virus (HCV) RdRP, which indicates that the protonated acidic residues are essential at the active site. Motif A is involved in the crucial process of sugar selection and magnesium coordination. All classes of polymerase have the conserved aspartate residue located at the end of the β-strand of this motif (Delarue et al., 1990). This residue is involved in coordination with divalent cation. Its mutation to cysteine or glycine leads to loss of activity as observed in case of HCV NS5B (Steitz, 1998) (Fig. 1.1B).

The structural motifs A and B are involved in nucleotide recognition and binding motif D in phosphoryl transfer and motif E in priming nucleotide-binding. Domain D also provides structural integrity to all RdRPs (Fig. 1.1C).

1.3.1 Structural Flexibility

The NTP recognition is an important event in the multistep process of RNA synthesis. It is a crucial step for correct NTP placement. It begins with binding of β-phosphate of NTP to the active site, resulting in its closure. Motif D, due to its flexibility, gets nearer to the β-phosphate group, which is one of the rate-limiting steps. The conformational changes in motif D act as selectivity factor and facilitate movement of other motifs (Yang et al., 2012). The other motif, i.e., motif B also undergoes minute movement, which helps it in template recognition and positioning of incoming NTP (due to the interaction between conserved residues on N-terminus of motif B and NTP). The flexibility of motif F contributes to intimate contact between positive charge bearing basic residues of motif F and NTP at the active site (Ding et al., 1998). The C-terminal aspartate residues of motif A and conserved asparagine residue of motif B help in selection of NTPs over dNTPs. These residues form hydrogen bonds with incoming NTP at 2ʹ-OH.

1.3.2 Divalent Metal Ions

It is proposed that two metal ions are important for the activity of the enzyme. One of the metal ions coordinates with α-phosphate group of NTP and the 3ʹ-OH of the nascent primer along with two consecutive aspartate residues in motifs C and A. The second metal ion coordinates with β- and γ-phosphate groups of NTP as well as with the first two aspartic acid residues of motif A and the first of the two consecutive Asp residues in motif C (Fig. 1.1D). The most common ions appear to be Mg²+ and Mn²+, while the other ions attenuate the enzyme activity. Bacteriophage φ6 RdRP, when complexed with Ca²+, resulted in inactive conformation.

1.4 Mechanism of Enzyme Action

1.4.1 Two Metal Ion Mechanism

Steitz proposed two metal ion mechanism based on the observation that exonuclease activity of DNA polymerase I in Escherichia coli was dependent on magnesium ions. The proposed mechanism involves initial attachment of magnesium ion to nucleotide forming complex at the active site. This is followed by binding of second metal ion with 3ʹ-OH group of the ribose sugar and α-phosphate group of incoming nucleotide. This metal ion helps in lowering of PKa of 3ʹ-OH group and facilitates nucleophilic attack of the hydroxyl group. Further, the β- and γ-phosphate groups are stabilized by negatively charged pentavalent phosphorous transition state (Steitz, 1993) (Fig. 1.1D).

1.4.2 Initiation of RNA Synthesis

There are two mechanisms observed for the synthesis of RNA by RdRPs: de novo (primer-independent) initiation and primer-dependent initiation mechanisms.

1.4.2.1 De Novo Initiation Mechanism

The de novo initiation mechanism involves a single nucleotide, which acts as a primer for the incoming nucleotides. This mechanism can be further classified into internal initiation mechanism and 3ʹ-terminal initiation mechanism.

The RdRP binds to 3ʹ end of template RNA followed by formation of initiation complex with NTP and an additional GTP. This complex is stabilized by some additional structures. Bacteriophage φ6 has a C-terminal domain with one tyrosine residue, which is involved in stabilization by piling with P-site of NTP (Butcher et al., 2001). This leads to polymerization reaction between first NTP and incoming NTP, resulting in subsequent release of GTP. Further, the RdRP translocates which is followed by subsequent polymerization reactions and elongation.

The other mechanism involves binding of 3ʹ end nucleotide of template RNA with cystine pocket. The first NTP arrives at +1 position, which is stabilized by Mn²+ ion followed by subsequent positioning and polymerization of next nucleotides (Te Velthuis, 2014).

1.4.2.2 Primer-Dependent Initiation

Various RdRPs also use a primer-dependent mechanism for initiation of transcription through protein primer, an oligonucleotide of defined origin having random sequence or back-primer initiation (Van Dijk et al., 2004). The primer mechanism involves the use of terminal 3ʹ-OH group, which is provided by protein. The first incoming nucleotide binds through phosphodiester bond to the residue of the protein, catalyzed by viral RdRP. Adenoviruses and picornaviruses encode a terminal protein (20 or more AAs) for this purpose (Choi, 2012). The dsRNA bacteriophage φ6 can replicate by back-initiation. In this method, 3ʹ end of the template RNA forms a small hairpin, which acts as primer-template to support the incoming nucleotide (Laurila et al., 2005).

1.5 Structures of Different Viral RdRPs

1.5.1 Hepatitis C Virus

Hepatitis C virus (HCV), a (+)-strand RNA virus, is the primary cause of liver disease across the globe. The majority of HCV acute infections are converted to chronic infections leading to the development of hepatitis in an average span of 20 years. The disease progresses into fibrosis of hepatic cells, consequent cirrhosis, leading to hepatic failure or development of hepatocellular carcinoma (Rubsamen-Waigmann et al., 2003). About 177.5 million people are affected with chronic HCV infection throughout the world (Petruzziello et al., 2016). The virus is transmitted through use of the infected needle, sexual contact, and from mother to child.

HCV nonstructural protein 5B (NS5B) is a 66 kDa membrane-associated protein, which is present at C-terminus (2420–3010) in its polyprotein. The HCV RdRP shares structural similarity with HIV RT having structurally equivalent palm and fingertips. The catalytic domain of HCV RdRP consists of 531 residues arranged in the form of thumb, fingers, and palm subdomains (Bressanelli et al., 1999). It contains 21 α-helices and 18 β-strands. The motif Gly-Asp-Asp present in NS5B is highly conserved in all RdRPs and is the central catalytic core of HCV (Fig. 1.2A and 1.2E). NS5B is host-independent enzyme, which can replicate the complete HCV sequence in in vitro (Yamashita et al., 1998).

Figure 1.2 (A) Structure of HCV RdRP representing various structural domains and active site (PDB–1CSJ); (B) structure of Poliovirus RdRP representing various structural domains (PDB–1RDR); (C) structure of DENV RdRP representing various structural domains and nuclear localization site (PDB–2J7U); and (D) structure of JEV RdRP representing various structural domains (PDB–4K6M) (E, F, G, H). Bar diagram representing the sequence and domain of HCV, Poliovirus, DENV, and JEV, respectively.

The thumb domain of NS5B consists of 370–531 amino acids of C-terminal of the polypeptide and contains 7 α-helices and 2 β-hairpins. The two repetition motifs of this polymerase share similarity with armadillo repeats that play an important role in protein–peptide/–protein recognition. The peptide segment between the thumb and palm domains shares similarity with the poliovirus. Each finger may be subdivided into proximal palm region having a cluster of α-helices folded together while fingertips form barrel-shaped distal portion. The 5ʹ end of template strand overhangs and enters the active site through cervice circumvented by fingers subdomain and loop spanning between thumb and fingers subdomains (Lesburg et al., 1999).

1.5.2 Poliovirus

Poliovirus is a human enterovirus belongs to family Picornaviridae. It is an RNA virus, which causes poliomyelitis. Poliovirus causes infection of central nervous system, which may lead to limb paralysis and also affect other body systems causing minor illness. Poliovirus spreads through fecal–oral route. It enters in the body via mouth, proliferates in intestinal cells, and is excreted in feces. It causes damage to neuron of motor cortex and brain stem as well as to anterior horn in spinal cortex, leading to paralysis of the hind limb (Yang et al., 1997). Polio virion is nonenveloped and consists of capsid made of four proteins VP1–4. It has an ssRNA having a positive polarity, which is made up of 7433 nucleotides with polyadenylated 3′ terminus and 5′ terminus covalently linked to a small protein (VPg) (Kitamura et al., 1981).

RdRP of poliovirus is 53 kDa macromolecule, which helps in RNA replication. It shares structural similarity with other viral RdRPs and has homology to right hand. It also contains one long open reading frame producing a polyprotein of molecular weight 247 kDa. The C-terminus 461 amino acid peptide of this polyprotein produces RdRP (Racaniello and Baltimore, 1981). This RdRP carries out replication of viral RNA (vRNA) in host cell cytoplasm producing genomic, messenger, and template RNA (Richards and Ehrenfeld, 1990).

The RdRP of poliovirus also consists of thumb, palm, and fingers. The thumb mainly consists of α-helix of C-terminus residues of palm. The top of thumb also interacts with the N-terminus of the polypeptide. The palm is composed of five strands namely A, B, C, D, and E, while the finger subdomain is composed of two sequences, polypeptide sequence that precedes motif A (residues 97–194) and small sequence between motifs A and B (residues 240–285). Apart from these structures, the polio RdRP also has unusual structural component composed of N-terminus residues of finger subdomain, which is not present in other RdRPs (Fig. 1.2B and 1.2F). The elemental structure of the palm subdomain is made up of two α-helices and a four-stranded β-sheet. The RdRPs of poliovirus and RT share similarity in top portions of the finger subdomains. The thumb subdomain of poliovirus RdRPs starts from β-7 strand, which interacts with β-5 and β-6 stands of motif E, forming a short three-stranded antiparallel β sheet. A series of five α-helices complete the thumb subdomain. The N-terminal of the RdRP plays an important role in the polymerase activity. For instance, deletion of residues 1–6 or Trp 5 causes complete loss of the polymerase activity (Plotch et al., 1989). The residues 12–25 are projected in the active site cleft and play an essential role in polymerization activity. Poliovirus replicates its RNA through membrane-associated replication complexes having the RdRP and various other viral proteins. Further, this complex may also have some host proteins required for replication (Baltimore et al., 1963; Caliguiri and Tamm, 1970).

1.5.3 Influenza A Virus

Influenza A virus (IAV) is a –ssRNA virus. It is responsible for human respiratory infections causing 2.5–5 million deaths every year (Heymann, 2014). The infection is more prevalent seasonal epidemic of winter’s in temperate regions and prominently present in tropical regions (Viboud et al., 2006). IAV is a segmented RNA virus having 6 segments referred to be vRNAs present inside a virion. They encode about 16 proteins through partially overlapping open reading frames and alternative splicing (Dubois et al., 2014). This RNA is double-stranded at 5ʹ end (last 13–14 nucleotides) also at 3ʹ end to a certain extent. These two ends share partial completory with each other and are bonded together by RdRPs (Robertson, 1979). Nucleoprotein monomers cover the single-stranded portion of these RNAs in sequence-independent manner. The viral ribonucleoprotein complex replicates the viral genetic information inside the host cell nucleus (Baudin et al., 1994). The RdRP binds to a cap-1 structure (m7GpppNm) of pre-mRNA and cleaves 10–15 nucleotides from this region, which leads to initiation of viral transcription (Fodor et al., 1994). The RdRP terminates its action, when reaches to an oligo-U tract that occurs just before the 5' end of the vRNA, followed by addition of a poly-A tail onto the viral mRNA (Poon et al., 1999).

The RdRP of IAV consists of three domains: polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2). The polymerase domain consists of less than 400 amino acid residues having motifs G, F, and A–E (from N- to C-termini) (Bruenn, 2003). It is one of the largest known RdRPs with a mass of approximately 250 kDa.

1.5.3.1 Polymerase Basic 1 Subdomain

It consists of 757 amino acid residues and made up of motifs A–F. It is the second largest domain of all three subunits. It is expressed in the mammalian targets and interacts with PA and PB2. This interaction is required for pppApG synthesis, which is the starting point of vRNA and cRNA synthesis. The small loop formed by the first 15 residues of PB1 helps in interaction with PA (Bruenn, 2003).

1.5.3.2 Polymerase Basic 2 Subdomain

The PB2 subdomain is divided into four domains. The N-terminal residues (1–35) are involved in PB1 binding. The C-terminal domain i.e., 627 domain (residues 535–684) is responsible for virulence.

1.5.3.3 Polymerase Acidic Subdomain

The PA subdomain digested with trypsin results in two fragments of 25 and 55 kDa connected with 20 amino acid residue linker. The C-terminus of PA subdomain interacts with N-terminus of PB1 (Guu et al., 2008). The W406, E410, G502, and E524 residues are involved in polymerase activity of PA domain (Fodor et al., 2002). The N-terminal cleavage fragment has endonuclease activity, which is responsible for cleavage of capped pre-mRNA or primer. The study of PA active site, cocrystallized with potential inhibitors, show the presence of numerous pockets in endonuclease domain, which can be used for the design of inhibitors (Kowalinski et al., 2012).

1.5.4 Dengue Virus

Dengue virus (DENV), a +ssRNA virus, belongs to Flaviviridae family. It is enveloped virus causing dengue fever in 50–100 million people every year (Rodenhuis-Zybert et al., 2010). The dengue fever is commonly known as backbone fever, dengue hemorrhagic fever, or dandy fever. It is predominant in tropical and subtropical countries and is transmitted through Aedes mosquito bite with an incubation period of 3–14 days and disease period of 3–7 days. The DENV has four different serotypes, i.e., DENV 1–4 having 60% genomic sequence identity, with approximately 90% sequence identity within a serotype (Blok, 1985).

The genetic constituent of DENV shows the presence of genomic RNA with type I methylguanosine cap at 5ʹ end and no polyadenylate tail. It is about 10.5 kb long and encodes a single polyprotein, which is further lysed into three structural and seven nonstructural proteins (Wengler and Wengler, 1981). DENV RdRP is the largest protein having a molecular mass of about 105 kDa sharing 67% sequence identity in all serotypes. The DENV RdRP also shows right-hand conformation. The RdRP has 27 α-helices and 7 β-strands. It has palm, thumb, and finger domains along with a nuclear localization site. The α-2 to α-4 helices of the thumb region and α-5 to α-7 helices of finger region constitute the nuclear localization site. This region is present between thumb and finger subdomains. The sequence 341-AMTDTTPFGQQRVFKEKVDT in the nuclear localization site forms a part of thumb subdomain, which is quite mobile. The residues 350–360, especially Arg352, Phe354, Glu356, and Lys357, present in 3–10 β3-helix, interact with the importin-β site, and help in translocation event to the nucleus. The residues 273–315, 416–496, and 543–600 constitute the finger subdomain of NS5. A concave surface is formed by solvent-exposed residues of helices α6, α14, and α15, which is present near the N-terminus of the RdRP. This region is also marked by the presence of two flexible loops L1 (309–321) and L2 (342–347), which helps in connecting the thumb and finger subdomains and also provides mobility to certain extent. The palm subdomain consists of residues 497–542 and 601–705, The core of the domain is formed by two antiparallel β-sheets (β4 and β5) surrounded by eight α-helices (α11–α13, α16–α20). It has four conserved motifs responsible for NTP binding and catalysis (Fig. 1.2C and 1.2G). The motif C bears active catalytic sequence site (GDD), having Asp663 and Asp664, which is located at the turn between strands β4 and β5. The subdomain also has the presence of Mg²+ ion near the catalytic site coordinated with motif A (Asp533) and motif C (Asp664).

The thumb subdomain is present near the end of C-terminus of the DENV RdRP (residues 706–900). The amino acid residues 782–809 form a priming loop, which partly occludes the active site. The DENV RdRP shows the presence of two zinc-binding sites: His712, His714, Cys728 of motif E and Cys446, Cys449, His441, and Glu437 and zinc ions may contribute to the structural stability (Yap et al., 2007).

1.5.5 Zika Virus

Zika virus (ZIKV), first emerged during the mid-20th century in Uganda and identified in humans in 1952. ZIKV disease is transmitted by the bite of Aedes mosquitoes. The disease prevails typically for 2–7 days. ZIKV infections may sometime result in Guillain–Barré syndrome in adults. It is an icosahedral, enveloped, nonsegmented +ssRNA virus (Faye et al., 2014). It has 11 kb long RNA having a 5ʹ cap structure and lacks a poly-A tail similar to DENV. Its genome, a long open reading frame, encodes three structural and seven nonstructural proteins in the form of polyprotein (Fields et al., 2007). The N-terminal portion of nonstructural protein 5 (NS5) has methyltransferase followed by RdRP separated by a small linker. The ZIKV RdRP shares high homology with RdRP of members of Flavivirus family with conserved motifs A–G.

The ZIKV RdRP consists of fingers subdomain spanning from α1–α9 and α12–β3 (residues 275–498 and 544–607, respectively), palm subdomain covering α10, α11, 310 helix η1 and α14 to 310 helix η2 (residues 499–543 and 608–709, respectively) and thumb subdomain spanning from β6 to α23 (residues 710–887). The active site is circumvented due to contact between fingers’ motifs F and G and thumb of the RdRP. The nuclear localization sequence (NLS) region of this RdRP is located between thumb and finger subdomains. The βNLS extends from α2–α3 (residues 316–367), and α/βNLS is present in the fingers and palm subdomains, i.e., helices α5, α6. The catalytic site is located in palm subdomain (β4, β5 strands) bearing active site residues, Asp665 and Asp666. Helices α19 and α20 form priming loop and are present in RNA-binding tunnel providing a closed conformation to RdRP (Lim et al., 2016). The ZIKV RdRP also shows the presence of two zinc-binding sites, one containing Glu439, His443, Cys448, and Cys 451 in finger subdomain and other containing His714, Cys730, and Cys849 along with a water molecule in thumb subdomain. The second zinc-binding site shares proximity with Ser712 and Arg731 and plays an important role in binding of incoming ribose nucleotides. The RNA in ZIKV RdRP is narrower in comparison to that in other members of the family. The two distinct drugable sites in ZIKV RdRP have been identified as first, the RNA template entry tunnel and second one situated near the active site in thumb subdomain (Noble et al., 2013; Noble et al., 2016).

1.5.6 Japanese Encephalitis Virus

Japanese Encephalitis Virus (JEV) causes Japanese encephalitis, a viral encephalitis affecting approximately 68,000 people every year. The Japanese encephalitis is prevalent in Asia Pacific region, especially southeast Asia and west Pacific region (Pan et al., 2011). The first outbreak of JEV was initially reported in Japan in the late 19th century. The disease is transmitted by Culex mosquito bite. Till now, there is no cure for the disease except the symptomatic treatments. JEV is a +ssRNA virus covered with viral capsid. It is an enveloped virus with protective antigen on its surface (He, 2006). The JEV induces ER stress, which triggers apoptosis through p38-dependent and CHOP-mediated pathways (Su et al., 2002). Its genome consists of 10,976 nucleotides, which encode as a single long open reading frame. It transcribes a long polyprotein, which is further cleaved to produce 3 structural and various nonstructural proteins (Sumiyoshi et al., 1987). The JEV NS5 contains MTase (residues 1–266, followed by residues 271–273) followed by JEV RdRP with a total of 905 residues (Fig. 1.2D and 1.2H).

The JEV RdRP comprises a central core, N-terminus extension (residues 276–303), and priming loop (residues 790–812). The core polymerase shows cupped right hand with a cavity surrounded by fingers and thumb. The palm subdomain contains two conserved active site residues, Asp536 (motif A) and Asp668 (motif C). The index fingertip and thumb interact with each other and form lining of the active site. The index finger also bears NLS (residues 322–370) and nuclear export signal (NES) (residues 329–345). The ring finger of JEV RdRP forms NTP entry channel roof and has NTP-binding site (motif F). The pinky finger, partially involved in dsRNA channel, contains motif G (residues 109–118) (Gong and Peersen, 2010). The priming loop has 23 amino acid residues (790–812), forming an insertion loop, which extends into the polymerase active site and plays an important role in de novo initiation process of transcription.

The JEV also has two zinc ions similar to other Flavivirus RdRPs. The first Zn²+ is present on pinky finger coordinated with Glu440, His444, Cys449, and Cys452. The other zinc ion is present between motif E and thumb domain. It is coordinated with water molecule and residues His717, Cys733, Glu848, and Cys852. The MTase and RdRP interact with each other by polar electrostatic interactions between the residues of ring finger Phe467, index finger Phe351 (βNLS core helix), middle finger Pro585 (RdRP) and the residues Pro113, Leu115, and Trp 121 (MTase) (Lu and Gong, 2013).

1.5.7 Human Rhinoviruses

Human rhinoviruses (HRVs), a group of three genetically different HRVs A, B, and C, cause common cold. They belong to Enterovirus genus and family Picornaviridae. The infection is spread through two modes: respiratory droplets and from fomites. The virus has an icosahedral structure with +ssRNA having approximately 7200 bp in its genome. The genome bears a single gene, which encodes 11 proteins, out of which four proteins, VP1–4, constitute the viral capsid. About 90% of serotypes of the virus use an intercellular adhesion molecule 1 (ICAM-1), a cell surface receptor for adhesion, while others use a low-density lipoprotein receptor (LDLR) (Jacobs et al., 2013).

The HRV genome encodes a polyprotein, whose C-terminal is cleaved to produce RdRP. The replication of genome occurs via primer-dependent mechanism in membranous vesicle of the cytoplasm of infected cells (Gohara et al., 2000). The HRV RdRP fingers subdomain has three regions, N-terminal segment (residues 1–54), inner region proximal to palm (residues 55–200), and distal region pointing away from palm (residues 243–290). The deletion of the first six residues of the RdRPs results in inactivation of the enzyme (Hobson et al., 2001). The inner finger regions are formed by α3, α9, α10, α13, and α14 helices, while the outer finger regions are made up of α5, α6, α7 helices, β1, β6, β8, β10, β11 sheets, β4–β5 hairpins, and a loop (residues 160–173).

The palm subdomain is formed of β-sheets, β9, β12, and β13 encircled by α11, α15, and α16. The motif A (α12, β9) of palm subdomain has Asp239, which helps in selecting ribosenucleotide over deoxyribosenucleotide. The motif B (α15) has conserved residue Asn296, which interacts with Asp239 and helps to position Asp239 for NTP recognition. The thumb subdomain consists of α17, α18, α19, α20, and α21 helices. The HRV RdRP has potassium-binding site at the boundary of inner and outer finger domains. Further, Hung et al. also observed that low potassium concentration provided a positive effect on enzyme activity. The potassium-binding site is about 15 Å away from the active site (Hung et al., 2002). The RdRP has conserved aspartic acid residues and is involved in the binding of two magnesium ions, where one ion helps in the action of primer’s 3ʹ-OH group and other stabilizes the phosphate groups. One of the magnesium ions is coordinated near Asp234, Asp327, Asp328, and Asp357 (motif C) while other is surrounded by Cys268, Lys267, Gly283, and Gly284 (Love et al., 2004).

1.6 Enzyme-Ligand Interaction Strategies (Drug Design and Development)

The enzymes are one of the important targets for the development of therapeutic agents. The identification of druggable target(s) followed by identifying possible modes of ligand–target interactions are important strategies in the process of drug design. The prediction of protein–ligand interactions through molecular docking involves utilization of existing protein–ligand interaction data and the whole process is started from the beginning (Rose et al., 2010). The existing protein–ligand complex models, elucidated through X-ray crystallography and NMR and now available in various open source protein databases, are gaining importance in strategies of drug design involving enzyme–ligand interaction (Konc et al., 2015). Further, the enzyme flexibility, solvent, and weak interaction are also taken into account (Engh et al., 1996). The transposition of ligand from a known protein–ligand complex to another protein having a similar binding site is another interesting strategy for drug design (Haupt et al., 2013). This technique is based on the assumption that three-dimensional accurate alignments of amino acid patterns or their corresponding functional groups in the proteins-binding sites is prerequisite for the ligand transposition.

The various approaches available for the inhibition of HCV RdRP include targeting the nucleoside-binding site (active site) or targeting the four allosteric sites through nonnucleoside inhibitors (Kwong et al., 2008). The scaffolds such as acrylic acids, benzodiazepine, benzothiadiazines, benzofurans, isothiazoles, pyrrolidines, rhodanines, and proline sulfonamides bind near the interface between palm and thumb subdomain (Vandyck et al.,