Norovirus RNA-dependent RNA-polymerase is phosphorylated 1

by an important survival kinase, Akt 2

John-Sebastian Eden†, Laura J. Sharpe†, Peter A. White* and Andrew J. Brown* 3

BABS, School of Biotechnology and Biomolecular Sciences, Biosciences Building D26, 4

University of New South Wales, Sydney, NSW 2052, Australia. 5

Running Title: Akt phosphorylates Norovirus RdRp 6

Keywords: Akt; Norovirus; RdRp; phosphorylation; kinase 7

Abstract word count: 97 8

Text word count: 1624 9

† These authors contributed equally to this work. 10

* These corresponding authors contributed equally to this work. 11

Mailing address: BABS, School of Biotechnology and Biomolecular Sciences, Biosciences 12

Building D26, University of New South Wales, Sydney, NSW 2052, Australia. 13

Fax: +61-2-9385-1483; 14

AJB: Phone: +61-2-9385-2005; E-mail: aj.brown@unsw.edu.au 15

PAW: Phone: +61-2-9385-3780; E-mail: p.white@unsw.edu.au 16

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.05562-11 JVI Accepts, published online ahead of print on 17 August 2011

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Abstract 17

Viruses commonly use their host cells’ survival mechanisms to their own advantage. We 18

showed that Akt, an important signalling kinase involved in cell survival, phosphorylates the 19

RNA-dependent RNA-polymerase (RdRp) from Norovirus, the major cause of gastroenteritis 20

outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the 21

more prevalent Norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs 22

at a residue (Thr33) located at the interface where the RdRp finger and thumb domains 23

interact, and decreases de novo activity of the polymerase. This finding provides fresh insights 24

into virus-host cell interactions. 25

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Norovirus (NoV) is the most common cause of acute gastroenteritis outbreaks, causing 28

vomiting and diarrhoea in affected individuals (14). Since 1996, five variants of the genogroup 29

II, genotype 4 (GII.4) lineage have been associated with global pandemics of acute 30

gastroenteritis of increased frequency (23). Overall, GII.4 variants are the cause of 62-80% of all 31

NoV outbreaks globally (11, 23). There are a number of factors contributing to this higher 32

epidemiological fitness including host-cell receptor binding patterns and duration of herd 33

immunity [reviewed in (7)]. Also, our group recently showed that the GII.4 viruses had a 34

greater capacity to evolve than other NoV genotypes through higher rates of mutation (6). 35

Therefore, the viral RNA-dependent RNA-polymerase (RdRp) is likely to play an important role 36

in driving viral evolution and fitness (6). Post-translational modifications are frequently found 37

on viral proteins (15), including RdRps (17), and this led us to examine a potential post-38

translational modification of this polymerase from a representative pandemic NoV strain, GII.4 39

2006b (NSW696T/06/AU - GenBank EF684915). This variant was associated with a global 40

pandemic in 2007-8 (23) and caused three consecutive epidemics of gastroenteritis in Australia 41

in 2006 (26), 2007 and 2008 (12). 42

Viruses take advantage of the host cells’ existing pathways and mechanisms, and one of 43

these is the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway (5). Akt is a 44

serine/threonine protein kinase that phosphorylates downstream targets leading to numerous 45

effects, with the net result of increasing cell growth, survival and proliferation. Consequently, 46

perturbations of the Akt signalling pathway have been implicated in the development of 47

diseases including many human cancers (2) and diabetes (28). With such a fundamental role in 48

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the regulation of cell growth and proliferation, it is perhaps not surprising that a number of 49

viruses have been found to interact directly with the PI3K/Akt pathway to effect greater control 50

of their replication within the host cell. In HCV, the expression of viral proteins including 51

NS3/4A, NS4B and NS5A result in the activation of the PI3K/Akt pathway [reviewed in (3)], 52

which is required for efficient virus replication (4). Similarly, Influenza A virus activates the 53

PI3K/Akt pathway through the viral non-structural protein 1 (13). This appears to suppress the 54

induction of apoptosis, which may arrest the cell at a stage that supports viral replication. 55

Alternatively, not all viruses activate the PI3K/Akt pathway. For example, in Measles virus, 56

infection leads to the down regulation of Akt that appears to plays a role in immune 57

suppression (8). 58

Given the common exploitation by viruses of Akt signalling, we sought to determine 59

whether the NoV RdRp may be a substrate for Akt. Akt phosphorylates proteins at the minimal 60

consensus sequence of RxRxx(S/T)h (where x is any amino acid, and h is a bulky hydrophobic 61

amino acid) (1). This minimal consensus motif was supported by another peptide library 62

screening approach (20). However, there is considerable variability from this consensus in the 63

sequences surrounding Akt phosphorylation sites (22). Therefore, to help determine the 64

potential for phosphorylation to occur on the NoV GII.4 2006b RdRp, we interrogated the 65

amino acid sequence using Scansite (21). This program predicts various phosphorylation sites 66

and protein binding motifs, including likelihood (stringency), based on minimal consensus 67

sequences. This indicated that the Thr33 residue of the 2006b RdRp was predicted as both a 68

high stringency Akt substrate site and 14-3-3 zeta binding site. The RdRp Thr33 residue is 69

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mostly conserved between pandemic strains, and almost absent in non-pandemic strains (Table 70

1, χ2: p<0.001). 71

In light of the in silico predictions and the complementary expertise of the investigators 72

(12, 22), we decided to confirm Akt mediated phosphorylation of purified recombinant RdRp. 73

Briefly, the coding region for NoV RdRp with a C-terminal hexahistidine tag was cloned into 74

pGEX-4T-1 (GE Healthcare), then expressed and purified as previously described (6). An N-75

terminal glutathione-S-transferase (GST) tag was included to increase the size of the RdRp to 76

allow separation of the RdRp away from the Akt protein, by SDS-PAGE. An in vitro Akt kinase 77

assay (22) was employed to compare wild-type RdRp (GII.4 2006b), with a mutated version – 78

Thr33Ala, where the predicted phosphorylation site had been mutated to alanine. To detect 79

phosphorylation, we made use of an antibody designed for identification of phosphorylated Akt 80

substrates [PAS, Cat #9611, Cell Signaling Technology, (29)]. Our results showed clear Akt-81

dependent phosphorylation of the wild-type GII.4 2006b RdRp (Figure 1), which was absent in 82

the mutated version. A second antibody that is more specific for phosphorylated Akt substrates 83

(Cat #9614, also from Cell Signaling Technology) also detected wild type phosphorylated RdRp 84

and not the mutated form (data not shown). In addition, we tested a representative RdRp with 85

a medium stringency site (GII.b C14) (C14/2002/AU - GenBank AY845056), and one with no 86

predicted phosphorylation site (GII.7 Mc17) (Mc17/2001/Th - GenBank AY237413). This 87

indicated that the medium stringency sequence was also phosphorylated at Thr33, but the GII.7 88

RdRp with no corresponding Thr residue was not (Figure 1). 89

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Akt’s phosphorylation of NoV RdRp may provide additional insights into the 90

evolutionary strategies by which viruses exploit the host for their own benefit. The Akt 91

pathway is active when cells are proliferating rapidly, so it is possible that NoV takes advantage 92

of this situation to increase its survival. How this phosphorylation event may influence the 93

function of RdRp is unclear, since there is currently very little information linking structure and 94

function of the NoV RdRp, largely due to the lack of a cell culture system for the virus. Most of 95

the important residues have been predicted from published crystal structures (19, 27) and 96

comparison of structures to functionally defined residues in Poliovirus and Foot and Mouth 97

Disease Virus [e.g. (25)]. The phosphorylated Thr33 residue is located at the tip of the finger 98

domain which interacts with the top of the thumb and encloses the active site (Figure 2A & B). 99

A study by Thompson et al. suggests that this structure, which is unique to RdRps, plays a role 100

in stabilising Poliovirus RdRp (25). These same residues may play a similar stabilising role in 101

NoV RdRp, allowing a conformation that facilitates RNA synthesis. Homology modelling 102

predicts extensive inter-domain interactions within this region of the RdRp including two polar 103

bridging interactions that link the finger and thumb domains at Thr33 in the GII.4 2006b RdRp 104

(Figure 2C). When the Thr33 residue is phosphorylated, the negative charge of the phosphate 105

should repel the negatively charged side chain of Glu468, altering the dynamics of these inter-106

domain finger/thumb interactions. 107

The HCV RdRp (NS5B) has been shown to be phosphorylated by Protein Kinase C-related 108

Kinase 2, and that phosphorylation event played a role in regulating HCV RNA replication (16). 109

To explore the possibility that the phosphorylation of Thr33 is functionally significant, we 110

compared the enzyme kinetics of the wild-type 2006b RdRp with a mutated form that should 111

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mimic phosphorylation (Thr33Glu), using a de novo GTP incorporation assay as previously 112

described (6). The phosphomimetic mutant had a lower maximum enzyme velocity (Vmax = 113

100 fmol.min-1) compared to the wild type RdRp (Vmax = 125 fmol.min-1), indicating a slower 114

rate of RNA synthesis (Figure 3). The phosphomimetic mutant also had a lower affinity for the 115

GTP substrate with a Km value of 10.4 µM, compared to 4.8 µM for the wild-type RdRp. The 116

differences in enzyme kinetics between the wild-type and phosphomimetic RdRp suggest that 117

the phosphorylation of Thr33 may modulate the function of the polymerase. In HCV, it has 118

been shown that disruptions to the interactions between the finger and thumb domains will 119

lead to a reduction in de novo RdRp activity, while efficient primer extension activity is 120

maintained (9, 10, 24). Since NoV utilises both de novo and protein primed RNA synthesis, it is 121

possible that phosphorylation of NoV RdRp could alter the balance between these modes of 122

RNA synthesis. 123

In contrast to examples of viral exploitation of the Akt pathway (3, 8, 13), a decrease in 124

polymerase activity could indicate that Akt phosphorylation of the NoV RdRp is an anti-viral 125

strategy by the infected cell. However, this may be hard to reconcile with our observation that 126

the Akt phosphorylation event occurs in the most epidemiologically fit genotype, GII.4 and is 127

generally absent from others. Further work will be needed to distinguish between these 128

possibilities and to determine the viral outcomes of this phosphorylation event. 129

Phosphorylation may not just alter function directly, but could act as a molecular signal 130

for a binding partner. In line with this, a high stringency 14-3-3 zeta binding site was predicted 131

by Scansite on the phosphorylated Thr33 residue. 14-3-3s are ubiquitous proteins that bind to 132

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phosphorylated residues; therefore further alterations of protein function could occur (18). In 133

this case, due to the high intrinsic disorder within this region of the polymerase, it is possible 134

that 14-3-3 binding may act as a clamp that fixes the polymerase into a functionally significant 135

conformation. The interaction of 14-3-3 zeta and NoV RdRp may also affect the localisation 136

within the cell to aid evasion of the host cell recognition system. The possibility that 137

phosphorylation of NoV RdRp may affect intermolecular interactions represents another 138

interesting avenue for further research. 139

Here, we have discovered a novel Akt substrate, a viral polymerase, and identified its 140

phosphorylation site. This residue appears to be critical for the activity of the NoV RdRp, based 141

on our in vitro polymerase assay. Importantly, it is routinely present in pandemic strains, but 142

mostly lacking in non-pandemic genotypes. Future work should include confirming the NoV 143

RdRp phosphorylation event in vivo, determining its impact on viral replication and whether 144

this favours the host or the virus. In this respect, it would be worthwhile to engineer the 145

phosphorylation site into the existing murine NoV infectious cell culture system. The current 146

work provides a good basis for future efforts to better understand how phosphorylation of viral 147

proteins may affect replication. 148

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Figure legends 236

Figure 1: Akt phosphorylates RdRp at Thr33 237

Wild-type (WT) or mutant (T33A) recombinant NoV GII.4 2006b, GII.b C14, or GII.7 Mc17 RdRp 238

(1 µg) were incubated with or without recombinant Akt (400 ng) as indicated at 30°C for 30 239

min. Reactions were stopped by addition of loading buffer, and samples were subjected to 240

SDS-PAGE and Western blotting with Phospho-Akt Substrates (PAS) antibody, followed by 241

stripping and probing for RdRp and Akt. Representative of three independent experiments. 242

The bold residue in the sequence indicates the residue corresponding to Thr33 in WT GII.4 243

2006b RdRp. Stringency values refer to Scansite stringency: <0.2 = high stringency and 0.2-1 = 244

medium stringency. 245

246

Figure 2: 3D-modelling of NoV RdRp showing finger-thumb domain interactions at Thr33 247

(A) Front view of the NoV GII.4 2006b RdRp highlighting the finger (red), thumb (blue) and palm 248

(yellow) domains. (B) Top view highlighting the fingertips (red) and C-terminal region (cyan). 249

(C) Close-up view of the boxed region of (B) showing the finger-thumb interactions at Thr33 for 250

2006b RdRp. Images produced using PyMOL, Version 1.3, Schrödinger, LLC. 251

252

253

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Figure 3: Comparison of enzyme kinetics of the wild-type 2006b RdRp with a Thr33Glu 255

phosphomimetic mutant 256

The enzyme kinetics of the wild-type 2006b RdRp (Wild) were compared with a Thr33Glu 257

phosphomimetic mutant (T33E). Briefly, 50 ng RdRp was added to initiate a reaction where 3H-258

GTP was incorporated onto homopolymeric C RNA. After a ten minute incubation, the reaction 259

was stopped, harvested onto a filterplate, and then the incorporated nucleotides detected via 260

scintillation counting. Samples were tested in triplicate and background values were subtracted 261

to obtain the measure of incorporated nucleotides as fmole.min-1. This was measured across a 262

range of GTP substrate concentrations, increasing to 51 µM. The data were analysed using the 263

Hill equation in GraphPad Prism, Version 5.04. The kinetic parameters are presented with 264

standard deviations. 265

266

Table 1 Footnotes 267

1RdRp residues 28 - 34 relative to GII4 2006b variant - NSW696T/06/AUS (GenBank Accession - 268

EF684915). The bold residue is the predicted phosphorylation site. 269

2Values refer to Scansite stringency: <0.2 = high stringency and 0.2-1 = medium stringency. 270

3Prevalence: ●●●●● indicates strain was associated with a global pandemic, ●●● indicates 271

strain was associated with a regional epidemic and ● indicates strain was associated with a local 272

outbreak or sporadic disease in the natural host. 273

*Strains used in Figure 1. 274

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