Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA Packaging Motors with

40 Pages·2016·4.5 MB·English

JVI Accepted Manuscript Posted Online 29 June 2016
J. Virol. doi:10.1128/JVI.00508-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA Packaging
2 Motors with Revolving Mechanism
3 Fengmei Pi1, Zhengyi Zhao1, Venkata Chelikani2, Kristine Yoder3, Mamuka Kvaratskhelia1, Peixuan Guo1*
4 1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy; Department of Physiology &
5 Cell Biology and Dorothy, M Davis Heart and Lung Research Institute, College of Medicine; The Ohio State
6 University, Columbus, OH 43210, USA. 2Faculty of Agriculture and Life Sciences, Lincoln University, PO Box D
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7 84, Lincoln 7647, Canterbury, New Zealand. 3Department of Molecular Virology, Immunology, and Medical nlo
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8 Genetics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. e
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9 Address correspondence to: r
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10 Peixuan Guo, Ph.D., Email: [email protected]; Phone: [phone] h
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11 Abstract :/
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12 The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate a
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13 development of new potent antiviral drugs. Recently, a method has been described for developing potent .o
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14 inhibitory drugs by targeting biological machines with high stoichiometry and a sequential action mechanism. o
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15 Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA packaging motors.
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16 Inhibiting multi-subunit targets with sequential action resembles breaking one bulb in a series of Christmas m
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17 lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development r

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18 of new antiviral drugs. Recent elucidation of the mechanism of the viral dsDNA packaging motor with ,
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19 sequential one-way revolving will promote the development of potent antiviral drugs with high specificity and 8

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20 efficiency. Traditionally, biomotors were classified into two categories: linear and rotation motion. Recently
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21 discovered was a third type of biomotor, including the viral DNA packaging motor, that use a revolving s
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22 mechanism without rotation. By analogy, rotation resembles the Earth rotating on its own axis, while revolving
23 resembles the Earth revolving around the Sun (see animations: http://nanobio.uky.edu/movie.html). Herein, we
24 review the structures of viral dsDNA packaging motors, the stoichiometry of the motor components, and the
25 motion mechanism of the motors. All viral dsDNA packaging motors contain a high stoichiometry machine
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26 composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent
27 drug development based on the power function of the stoichiometry of target complexes that work sequentially.
28 Introduction
29 Viruses reproduce themselves in host cells. Their intracellular parasitic nature poses a great challenge to
30 antiviral drug development. Nevertheless, significant progresses have been made in antiviral drug discovery in
31 the past, such as new treatments for HIV(1), Hepatitis B virus(2), herpes virus (3) and influenza virus(4). Even D
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32 though different approaches for new drug development have been explored, such as improving drug target n
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33 binding affinity(5) and finding new targets with novel pharmacological mechanisms(6), new infectious d
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34 pandemic viruses still emerge sporadically that greatly threaten human health. In addition, the emergence of f
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35 drug resistance necessitates new drug development. Highly potent drugs with elevated specificity are needed for
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36 overcoming viral diseases. :
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37 In viral reproduction, the key step of genome packaging is usually accomplished by a biomotor using i.a
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38 ATP. Linear dsDNA or dsRNA viruses package their genomes into preformed procapsids. This group includes .
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39 dsDNA/dsRNA bacteriophages(7), adenoviruses(8), poxviruses(9), human cytomegaloviruses (HCMV)(10), /

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40 and herpes simplex viruses (HSV)(11). Intrigued by the unique viral structure and packaging mechanisms, N
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41 extensive studies have been carried out to elucidate the fundamentals in protein/DNA, protein/RNA and e
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42 RNA/DNA interactions in the quest for new prototypes of biological machines or new antiviral drugs. Jonathon e
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43 King’s observation several decades ago that studies on viral DNA packaging will lead to the discovery of new ,

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44 antiviral drugs(12) has stimulated scientists to pursue further study on the viral DNA packaging mechanism 1
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45 with the goal of uncovering better drug targets. Indeed, our study on phi29 packaging motor revealed that, y

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46 several key motor components composed of multimeric complexes, such as hexameric pRNA ring, hexameric e
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47 gp16 ATPase, and ATP with more than 10,000 copies to package one genome(13,14), may serve as better drug
48 targets than a monomeric genomic DNA. Comparing the viral packaging inhibition efficiency of mock drugs
49 targeting each machine with different stoichiometry, we found that inhibition efficiency increased exponentially
50 to the stoichiometry of the targeted bio-complex (Fig. 1A)(14).
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51 Previously, biomotors were classified into two categories: linear and rotation motors(15). Recently, a
52 new category of biomotors using a revolving mechanism without rotation was reported and found to be
53 widespread in different biological systems (Fig.1B)(16,17). Based on this new mechanism, a method for
54 developing highly efficient inhibitory drugs was described by targeting biological machines with high
55 stoichiometry and exhibiting sequential action mechanism(14,18). Herein, we review the development of
56 antiviral drugs that target viral DNA packaging motors, a subject that has been investigated for many decades. D
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57 A new method for developing highly potent drugs by targeting homomeric biological n
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58 machines with high stoichiometry to act sequentially d
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59 A determining factor for the drug inhibition efficiency on its target biological entity is the ratio of the r
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60 drugged (T ) to undrugged (T ) target components. Within each viral infected cell, a higher percentage h
inactive active
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61 of drugged machines (T ) leads to higher inhibitory efficiency. The percentage of drugged machines can be :/
inactive /
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62 calculated from binomial distribution or Yang Hui’s Triangle (Fig. 1C): a
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63 (cid:4666)(cid:1868)+(cid:1869)(cid:4667)(cid:3027) =(cid:3435)(cid:3027)(cid:3439)(cid:1868)(cid:3027)+(cid:3435)(cid:3027)(cid:3439)(cid:1868)(cid:3027)(cid:2879)(cid:2869)(cid:1869)(cid:2869)+⋯(cid:3435) (cid:3027) (cid:3439)(cid:1868)(cid:2869)(cid:1869)(cid:3027)(cid:2879)(cid:2869)+(cid:3435)(cid:3027)(cid:3439)(cid:1869)(cid:3027) =(cid:3533)(cid:3027) (cid:3435)(cid:3027)(cid:3439)(cid:1868)(cid:3014)(cid:1869)(cid:3027)(cid:2879)(cid:3014) (Equation 1). Here, p and q .or
(cid:2868) (cid:2869) (cid:3027)(cid:2879)(cid:2869) (cid:3027) (cid:3014) g
(cid:3014)(cid:2880)(cid:2868) /

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64 represent the fraction of drugged inactive and non-drugged active subunits in the population, Z is the
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65 stoichiometry of the target, and M represents drugged subunits in each biocomplex. v
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66 1) The first intrinsic factor of the target for potent drug development is Z, which is the stoichiometry of b
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67 the homomeric complex serving as a drug target. The ratio of functional complexes changes dramatically with Z. 7
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68 In equation 1, assuming that the ratio of drugged inactive subunits (p) and undrugged active subunits (q) in the 0
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69 population is fixed, when the target machine contains only one subunit, the ratio equals to q as derived from b
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70 Z=1 in binomial distribution (Equation 2). Here p + q = 100%. u
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71 (cid:4666)(cid:1868)+(cid:1869)(cid:4667)(cid:2869) =(cid:1868)+(cid:1869) (Equation 2)
72 However, when the homomeric target complex contains multiple subunits, a binomial distribution
73 formula with a higher order (Equation 3) is applied. When Z=4, then:
74 (cid:4666)(cid:1868)+(cid:1869)(cid:4667)(cid:2872) =(cid:1868)(cid:2872)+4(cid:1868)(cid:2871)(cid:1869)(cid:2869)+6(cid:1868)(cid:2870)(cid:1869)(cid:2870)+4(cid:1868)(cid:2869)(cid:1869)(cid:2871)+(cid:1869)(cid:2872) (Equation 3)
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75 In this case, the probability of a target machine complex possessing four copies of the inactive subunit is
76 p4; three copies of the inactive and one copy of the active subunit is 4p3q; two copies of the inactive and two
77 copies of the uninhibited subunits is 6p2q2; three copies of the active subunits is 4pq3; and four copies of the
78 active subunit is q4. Assuming that 70% (p) of subunits are inactivated by drugs, the percentage of active
79 machines containing four copies of active subunits is q4, which is (0.3)4 = 0.8%. However, when Z=1 and p=70,
80 the ratio of the uninhibited portion equals to 30%. Thus, the stoichiometry (Z) of the targeted machine D
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81 contributes significantly to the ratio of survival rate, which directly correlates with drug inhibition efficiency on
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82 viral replication.
d
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83 2) The second intrinsic factor of the target for potent drug development is K, which is the number of
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84 drugged subunits required to block the function of the complex. Stoichiometry only has a multiplicative effect

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85 on inhibition efficiency when K=1. Reinterpreting this statement with an example of a homo-hexamer machine p
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86 (Z=6) as a drug target, where 70% (p) of subunits are inactivated by drugs: if K=1, the ratio of active target i.
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87 complexes will be 0.36=0.07%; whereas if K=6 the ratio will be 1-0.76=88.2%. K=1 showed much stronger m
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88 inhibition compared to the case of K=6, where the active complex ratio was 88.2% at the same blocking subunit g
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89 ratio (Fig.1D). K=1 is a key factor for multisubunit complexes as potent drug targets.
N
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90 3) The third intrinsic factor is that the homomeric complex acts through a sequential or coordination e
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91 mechanism. Sequential or coordination action means that each subunit of the complex works in turn to complete e
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92 the function of the complex (Fig.1E). Blocking any step of the sequential action results in deactivation of the 7
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93 complex. That meets the definition of K=1 in a homomeric complex. Analogous to a string of Christmas lights,
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94 where one broken light bulb will turn off the entire chain, one inhibited subunit will deactivate the entire b
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95 complex and consequently their biological activity.
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96 The packaging motors of dsDNA viruses meet the first intrinsic factor of the target for
97 potent drug development as homomeric multimers with high stoichiometry
98 Viral DNA packaging can be generally divided into two closely related categories(19): (1) terminase-
99 portal channel mediated DNA translocation, as in herpes viruses (20), adenoviruses (8) and many
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100 bacteriophages including phi29(21), T4(22), T3(23), T5(24), T7(25), λ(26), SPP1(27), HK97 (16,28); and (2)
101 HerA/FtsK type translocase, as in poxvirus, and other nucleocytoplasmic large DNA viruses (NCLDV)(29,30).
102 Both categories use a very similar revolving mechanism(13,15,21,28). The first category of viral DNA
103 packaging motors uses a pair of DNA packaging enzymes, a head and tail connector with high stoichiometry for
104 DNA packaging activity. For example, the bacterial virus λ motor contains a tetramer terminase composed of
105 two subunits of the large ATPase gpA and two subunits of gpNul1(31). The phi29 motor contains a hexameric D
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106 packaging RNA ring(32-34) and a hexameric ATPase gp16(35). The connector of this group of viruses is a
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107 homomeric dodecameric complex. Both the terminase and the connector meet the first requirement: homomeric
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108 complex with a high stoichiometry.
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109 The phi29 motor is the most well-studied model system for viral DNA packaging, the components of

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110 which were used as mock drug targets in recent studies to compare the influence of target stoichiometry on drug p
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111 potency(14). In 1998, the packaging RNA (pRNA) ring in the phi29 motor was first determined to be a i.
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112 hexamer(32,33) (featured by Cell (34)) (Fig.2A). Hexameric pRNA formation was verified by cryo-electron
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113 microscopy (Cryo-EM)(36), biochemical analysis(32-34), single molecule photobleaching assay(37), gold
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114 labeling imaging by electron microscopy (EM)(38,39), and RNA crystal structural studies(40). Recent
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115 experimental evidences based on binomial distribution analysis, qualitative DNA binding assays, capillary e
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116 electrophoresis and electrophoretic mobility shift assays(35,41,42) revealed that the ATPase gp16 of the phi29 e
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117 motor, which belongs to the ASCE superfamily, is a hexamer in its final oligomeric state(35), similar to the 7
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118 oligomeric ATPase ring of many other DNA translocases(43-45). Similarly, in the FtsK motor (Fig.2B), the α
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119 and β domains assembled into a hexameric ring-shaped multimer with a central channel through which the b
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120 dsDNA substrate is translocated (46). All these high stoichiometric multimeric complexes in dsDNA packaging
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121 motors fit the requirement on target for potent drug development.
122 Adenoviruses (AdV) are a group of well-studied dsDNA viruses that infect eukaryotic cells in vertebrates
123 including humans. AdV packages the genome with two subunits of the terminal proteins into capsids that
124 contain hexon, penton, and fiber(47). Proteins Iva2 and L1 52/55K are AdV packaging proteins. It has been
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125 reported that higher-order IVa2-containing complexes formed on adjacent packaging repeats are required for
126 packaging activity(48). Quantitative mass spectrometry, metabolic labeling, and Western blot revealed that
127 there are approximately six to eight IVa2 molecules in each particle (47). The high-order Iva2 motor protein
128 complex meets the first requirement of a high stoichiometry target for potent drug development.
129 Herpes simplex viruses (HSV) package their dsDNA genome into preformed protein shells using
130 terminases(49), which contain a large subunit pUL15 and a small subunit pUL28(50). pUL15 cleaves D
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131 concatemeric viral DNA during packaging initiation and completion cycles and functions as an ATPase
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132 providing energy to the packaging process. X-ray structure of the C-terminal domain of pUL15 showed a homo-
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133 trimer structure (Fig. 2C)(51), thus fitting the characteristics of high stoichiometry homomeric complex for high
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134 potent drugs.

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135 Mimivirus, Megavirus, Pandoravirus and Pithovirus(52-54) all belong to the nucleocytoplasmic large DNA p
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136 viruses (NCLDV) superfamily and infect a wide range of eukaryotes(55,56). Nine genes that are shared by all i.
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137 NCLDV families have been identified to encode for DNA polymerase: a capsid protein, 3 helicases, a virion
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138 packaging ATPase, a thiol oxido-reductase, a protein kinase, and a transcription factor (Fig. 2D)(57).
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139 Mimiviruses package their 1.2 Mbp dsDNA genome into preformed procapsids through a non-vertex portal(58)
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140 driven by the vaccinia virus A32-Type viron packaging ATPase (59). It has been shown that the structure and e
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141 function of their DNA packaging are homologous to the FtsK DNA translocase(55). e
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142 Poxviruses are large brick-shaped dsDNA viruses that replicate in the cytoplasm of infected cells. The two 7
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143 DNA strands of the genome are connected at the ends through hairpin termini(60). Poxvirus ATPase is coded
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144 by the A32 gene. Comparative sequence analysis revealed highly conserved N-terminal region with five motifs b
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145 among all poxviruses, including ATPase feature Walker A and Walker B motifs, A32L specific motifs III and
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146 IV, and a novel motif-V. The secondary structure predictions of the N-terminus of the A32 ATPase protein are
147 homologous to the FtsK DNA translocase(19).
148 The packaging motors of dsDNA viruses meet the second intrinsic factor of the target for
149 potent drug development with K = 1, as one inactive subunit blocks the entire machine
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150 The K value is a key factor in estimating the probability of inactive nanomachines or biocomplexes by
151 combination and permutation analysis. When K is 1, the uninhibited biocomplexes will equal to qz. Thus,
152 stoichiometry has a multiplicative effect on inhibition. Most multi-subunit homomeric biological motors act
153 through sequential coordination mechanisms, where blocking one subunit of the complex inhibits the function
154 of the whole nanomachine. Nucleic acid translocation and duplex unwinding by helicases are coupled to the
155 NTP binding and hydrolysis cycle. This represents an extremely high level of coordination between proteins D
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156 with their DNA substrates. ATPase undergoes conformational entropy changes upon ATP binding or ATP
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157 hydrolysis, leading to a respective high or low binding affinity towards DNA(16,35,61).
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158 Sequential action of the phi29 DNA packaging motor was reported in 1997(61,62) by Hill constant
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159 determination and inhibition assay using a binomial distribution (16,17). ATPase activity has been analyzed by

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160 studying the effects of introducing mutant gp16 subunits (41,42). The cooperative profile of subunits inside the p
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161 ATPase hexamer mostly overlapped with a model where one single inactive subunit is able to inactivate the i.
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162 whole oligomer. The asymmetrical ATPase structure has been widely observed, where oligomeric ATPase
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163 works through sequential binding and hydrolyzing ATP to push dsDNA translocation; the subunit of ATPase in g
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164 ATP bound state is shown as asymmetric in EM images(63). Thus the hexameric ATPases in the DNA
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165 packaging motor fit the mathematical model of K=1. e
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166 In AdV, both L4-22K(64) and L4-33K proteins(65) play important roles in virus assembly(66-68). e
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167 Removal of the C-terminal 47 amino acids of L4-33K aborted viral assembly function(67). L4-33K also plays a 7
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168 role in bovine AdV assembly(69). Similarly, removal of the 87 amino acids of bovine AdV 33K completely
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169 blocked capsid assembly(69). It has been reported that L4-33K is a virus-encoded alternative RNA splicing b
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170 factor and is also involved in viral DNA packaging(65). The presence of IVa2 protein as a hexamer/octamer
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171 complex at the unique vertex has been demonstrated and reported to be equivalent to packaging ATPase(47).
172 This assembly process is analogous to common features of portal-translocase systems. The ATPase property of
173 Iva2 protein and oligomeric structures found in the viron vertex indicate that they work cooperatively as
174 ATPases for packaging machinery, which leads to K=1. The major protein involved in poxvirus DNA
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175 packaging is the gene product of A32, which is also an ATPase. This ATPase has sequence similarity to the
176 products of the IVa2 gene of AdV, which is also an ATPase involved in DNA packaging(70).
177 HSV uses more than seven viral proteins (encoded by UL6, UL15, UL17, UL25, UL28, UL32, and UL33)
178 for the cleavage and packaging of its genome into procapsids (50). The terminase is comprised of the UL15,
179 UL28, and UL33 proteins, which act for DNA packaging (50,71,72). The structure of the pUL15 nuclease
180 domain strongly suggests an evolutionary path from simple phages such as siphoviruses to complex phages and D
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181 eventually eukaryotic herpes viruses(51). The structure of C-terminal trimeric pUL15 is easily superimposable
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182 with those of phage terminase large-subunit nuclease domains, and also superimposes well onto the HCMV
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183 pUL89C(51,73). Also, the full length protein might be a larger multimer with the motor function of the protein.
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184 These evidences suggest that the trimeric pUL15 functions cooperatively for its ATPase activity, and one

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185 blocked subunit could deactivate the whole multimer. p
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186 Mimivirus and other DNA packaging ATPase motors of NCLDVs and prokaryotic viruses with an inner i.
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187 lipid membrane belong to the FtsK/SpoIIIE/HerA superfamily (ATPases of bacteria and Archea)(15,28,74). An
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188 automated homology model of packaging ATPase has been predicted using bioinformatics tools in vaccinia g
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189 virus(19). These packaging ATPases possess conserved domain in addition to Walker A and Walker B domains.
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190 Blocking one component of genome packaging machinery might lead to disruption of all genome packaging in e
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191 these viruses. The blocking agents/drugs targeting genome components in NCLDVS can also be employed in e
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192 prokaryotes, as they have been predicted to use a similar revolving mechanism for genome packaging 7
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193 (15,28,55,75). Inhibitors targeting the genome packaging components of NCLDVs might even act as potential
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194 anti-bacterial drugs. This research becomes even more important as new antibiotic resistant strains are b
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195 emerging.
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196 The DNA packaging motor of dsDNA viruses meets the third intrinsic factor of the target
197 for potent drugs: sequential or coordinated action of homomeric biomachines
198 One key aspect of targeting homomeric biomolecules of viral packaging motors for drug discovery is that
199 the multi-subunit biological complexes follow a sequential coordination or cooperative mechanism(16,62,76-
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200 82), which is widely seen in biological systems(76,82,83). Inhibiting any subunit of the motor with sequential
201 action leads to deactivation of the entire system.
202 Motors of dsDNA viruses share a common revolving mechanism without rotation (Fig.3A)(17). Subunits of
203 these motors coordinate with each other forming a special structure to drive dsDNA translation through a one-
204 way traffic mechanism with high efficiency (84). Revolving motors can be distinguished from rotation motors
205 by channel size, as all revolving motors have a smaller channel size to allow the bolt and nut to contact, but the D
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206 channel size of revolving motors are bigger to allow the dsDNA to revolve (Fig. 3B). The 30° left-handed twist
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207 of the channel wall produces an anti-parallel arrangement with the right-handed helix of the dsDNA(16,84). The
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208 anti-chiral property between the left-handed twist of channel protein with the right-handed dsDNA can be a
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209 characteristic for revolving motors, as rotation motors have right-handed channels for DNA translocation(Fig.

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210 3C)(16,17). The electropositive lysine layers in the motor channels interact with a single strand of the p
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211 electronegative dsDNA phosphate backbone, resulting in a relaying contact that facilitates one-way motion and i.
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212 generation of transitional pauses during dsDNA translocation(84,85).
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213 Protein molecular motors act as switches that undergo conformational changes depending on the binding g
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214 of nucleotide. For example, in the hexameric ring of gp16 ATPase subunits of the phi29 DNA packaging motor,
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215 the ATP bound subunit has high affinity to dsDNA. Hydrolysis of ATP to ADP generates a conformational e
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216 change in the subunit resulting low affinity to dsDNA. This reduced affinity allows the adjacent ATP-bound e
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217 subunit to bind the dsDNA, thus translocating the dsDNA forward. Sequential firing of subunits around the 7
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218 hexameric ring has been further reported to be regulated by Arginine finger, leading to highly processive
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219 movement of the viral dsDNA into the phage capsid (63). b
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220 The main motor protein Iva2 in AdV is multifunctional. It assists in the assembly of the capsid and
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221 activates late transcription. Comparing the IVa2 protein sequences with ATPase from different species revealed
222 conserved Walker A and B motifs associated with binding and hydrolysis of ATP(86). Similar to the ATPases
223 in bacteriophage packaging motors, the multimeric ATPase IVa2 motor protein complex also works through a
224 sequential action to provide energy for packaging DNA into their capsids. IVa2 also interacts with a viral L4-
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225 22K protein, which is involved in genome encapsidation (87-89). The stoichiometry of IVa2 has been found to
226 be equal for different forms of assembly intermediates such as the empty capsids, light intermediate particles,
227 and unmatured and matured virions(90). IVa2 mutants were defective in DNA packaging and resulted in
228 accumulation of empty capsids similar to the procapsid of dsDNA bacteriophages(47).
229 Herpes virus terminase contains a large subunit pUL15, which cleaves concatemeric viral DNA during
230 packaging initiation and completion. The structure of the C terminal domain of pUL15 resembles that of D
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231 bacteriophage terminases, RNase H, integrases, DNA polymerases, and topoisomerases, with an active site
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232 clustered with acidic residues. The DNA-binding surface is surrounded by flexible loops, indicating that they
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233 adopt conformational changes upon DNA binding(51). These conformational changes are similar to the
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234 sequential action of ATPase gp16 observed in phi29 DNA packaging motors, which provides energy to support

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235 the one-way traffic of genomes into procapsids. p
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236 Recent microscopic studies provided insight into genome packaging in Mimivirus(55,58). DNA is i.
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237 packaged into preformed procapsids through a non-vertex portal site which is distal to the DNA delivery site.
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238 As described earlier, the viral packaging pathway in Mimivirus is similar to chromosome segregation in bacteria
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239 and requires interaction of the packaging motor with recombinases and topoisomerases(55). It has been
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240 proposed that the DNA entry portal is closed once genome packaging is complete, similar to the ATPase e
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241 SpoIIIE which promotes membrane fusion after bacterial DNA segregation(91). Interestingly, NCLDV e
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242 members such as Marseillevirus and Lausannevirus encode histone like proteins (92,93). Genome condensation 7
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243 becomes essential with large genome size(92), and these condensing proteins (Histone like proteins) may be
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244 required in Mimivirus to package its large genome (Fig. 3D). These proteins are present in most prokaryotes b
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245 and Eukaryotes for genome condensation (94-96).
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246 Perspectives
247 In the virology community, significant efforts have been made in the quest for antiviral drugs focusing
248 on identification of targets for drugs with high specificity. But little attention has been paid to finding new
249 methods for antiviral drug development. Above we reviewed a new methodology for anti-viral drug
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