Advances Toward a Norovirus Antiviral: From Classical Inhibitors to Lethal Mutagenesis

Abstract

Human noroviruses are a leading cause of gastroenteritis worldwide, yet there are no licensed antivirals. There is an urgent
need for norovirus therapeutics, particularly for chronic infections in immunocompromised individuals, but also a potential
need for prophylactic use in epidemics. Continued research has led to the identification of compounds that inhibit norovirus
replication in vitro and, at least in some cases, are also effective in vivo against murine norovirus. Progress has included
classical approaches targeting viral proteins and harnessing the antiviral action of interferon, strategies targeting essential
host cell factors, and novel strategies exploiting the high mutation rate of noroviruses.

Full text

The Journal of Infectious Diseases
SUPPLEMENT ARTICLE
Advances Toward a Norovirus Antiviral: From Classical
Inhibitors to Lethal Mutagenesis
Lucy Thorne,
a
Armando Arias,
a
and Ian Goodfellow
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, United Kingdom
Human noroviruses are a leadi ng cause of gastroenteritis worldwide, yet there are no licensed antivirals. There is an urgent need for
norovirus therapeutics, particularly for chronic infections in immunocompromised individuals, but also a potential need for pro-
phylactic use in epidemics. Continued research has led to the identification of compounds that inhibit norovirus replication in vitro
and, at least in some cases, are also effective in vivo against murine norovirus. Progress has included classical approaches targeting
viral proteins and harnessing the antiviral action of interferon, strategies targeting essential host cell factors, and novel strategies
exploiting the high mutation rate of noroviruses.
Keywords. human norovirus; antivirals; protease; polymerase; interferon λ; favipiravir; lethal mutagenesis.
Human noroviruses (HuNoVs) are a major cause of viral gas-
troenteritis worldwide, and since introduction of the rotavirus
vaccine they have become the leading cause of severe pediatric
gast roenterit is [1]. HuNoVs present a particular problem in
healthcare settings, with close to 4000 hospital outbreaks re-
ported in a 2-year period in the United Kingdom, causing ap-
proximately 9000 days of ward closures, disrupted services, and
a significant econo mic cost for the UK National Health Service
[2]. Most infections are acute with a low risk of mortality, but
HuNoV is recognized as a significant risk factor for compli-
cations and increased mortality in immunocompromised indi-
viduals, including transplant recipients and those receiving
immunosuppressive therapies [3]. In man y cases, HuNoV in-
fections in these cohorts can persist for years [4]. Conservative
estimates of mortality rates in develo ping cou ntries are much
higher ; at least 200 000 deat hs per year in children aged <5
years are attributed to HuNoVs [5].
Despite an urgent need, there are currently no licensed anti-
virals or vaccines for HuNoVs. Groups that would most benefit
include high-risk populations, such as young and elderly indi-
viduals, chronically infected patients, and medical staff. Health-
care and military settings could also benefit from prophylactic
use of antivi rals to potentially contain epidemics. However, the
development of therapeutics has been hindered by the lack of an
efficient cell culture system for HuNoV, which has also slowed
elucidation of the molecular details of noroviru s replication and
viral protein functions, key knowledge for the rational develop-
ment of specific antivirals. A replicon system, in which cells har-
bor self-replicating RNA of the prototype HuNoV, namely
Norwalk virus, has provided an invaluab le tool to evaluate an-
tivirals in vitro [6]. In the past few years, however, there has been
significant progress in the field, with the establishment of a plas-
mid-based reverse genetics s y s tem, to allow production of genet-
ically defined HuNoVs, and the first demonstration of HuNoV
replication in vitro in a cultured B-cell line and in vivo in immu-
nodeficient mice, albeit with limited replication in both [7–9].
These developments may now provide systems to evaluate poten-
tial antivirals, but their usefulness will critically depend on being
robust and reproducible in other laboratories.
Murine norovirus (MNV), which replicates in cultured mac-
rophage and dendritic cells, has been used as a surrogate model
for HuNoV since its discovery in 2003, owing to the availability
of reverse genetics systems and small-animal models. Both
HuNoV and MNV belong to the Caliciviridae family of small,
positive-sense, single-stranded RNA viruses and have similar
genome organization, protein functions, and conserved molec-
ular me chanisms of genome replication and translation. By use
of a combination of MNV and the HuNoV replicon, research
efforts into antivirals have intensified in the last 10 years [6,
10]. This review will focus on the latest developments, which
can be divided into categories of classical antiviral approaches
that target viral proteins or harness the antiviral effects of inter-
feron (IFN), alternative strategies targeting ho st cell processes,
and strategies that exploit the high mutation rate of noroviruses
by lethal mutagenesis.
DEVELOPMENTS IN CLASSICAL ANTIVIRAL
APPROACHES: RENEWE D INTEREST IN IFN AND THE
TARGETING OF VIRAL PROTEINS
Type I and type II IFNs elicit effective antiviral responses
against HuNoV and MNV, emphasizing the critical role of
a
L. T. and A. A. contributed equally to this work.
Correspondence: L. Thorne, Division of Virology, Department of Pathology, University of Cam-
bridge, Addenbrookes Hospital, Hills Rd, Cambridge, CB2 2QQ, UK (lt375@cam.ac.uk).
The Journal of Infectious Diseases
®
2016;213(S1):S27–31
© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of
America. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly
cited. DOI: 10.1093/infdis/jiv280
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innate immunity in controlling norovirus infections [6, 11]. De-
spite this, clinical use of IFN against HuNoVs has not been de-
scribed. Interest in IFN a s a HuNoV therapeutic has recently
been renewed, owing to a study that found that type III IFN,
IFN-λ, is required to control persistent MNV infecti ons [11].
Treatment with IFN-λ cleared persistent infections in mice
without requiring an adaptive immune response, revealing the
potential of IFN-λ as a treatment for chronic infections in the
immunocompromised.
A classical antiviral strategy is to target e ssential viral pro-
teins, and for HuNoV the viral protease (NS6
pro
) has become
the most widely studied antiviral target [10]. NS6
pro
is a chymo-
trypsin-like cysteine protease responsible for cleavage of the
viral polyprotein to release mature forms of the essential non-
structural replicase proteins, including itself (Figure 1). Resolu-
tion of the NS6
pro
crystal structure in 2006 has since facilitated
structure-guided design of a variety of inhibitors aimed to
mimic natural substrate recognition and react irreversibly
with active site residues. In th e past 2 years, t here has been a
significant increase in the number of norovirus protease inhib-
itors that exhibit a range of potencies in in vitro enzymatic as-
says and cell-based assays [10]. NS6
pro
shares similarities with
the picornavirus protease (3C
pro
), and some compounds effec-
tive against 3C
pro
exert broad reactivity against NS6
pro
.Most
recently, rupintrivir, originally developed against the rhinovirus
3C
pro
, was found to clear cells of HuNoV replicon RNA and in-
hibit MNV replication in vitro. In enzymatic assays, rupintrivir
inhibited the NS6
pro
of the predominant circulating HuNoV ge-
notype GII.4, suggesting that it may target clinically relevant
strains [12].
Given their essential role in replicating the viral genome, viral
RNA–dependent R NA polymerases (RdRps) also present at-
tractive antiviral targets. Polymerase inhibitors have been clin-
ically approved for many RNA viruses, whose RdRps share
conserved structural and functional properties with the norovi-
rus RdRp [13]. RdRp inhibitors can be divided into nucleoside
analogues, which target the active site, and non-nucleoside an-
alogues. The nucleoside analogue 2′ -C -methylcytidine
(2CMC), originally developed for use against hepatitis C virus
(HCV), inhibits HuNoV replication in vitro, and treatment with
2CMC cleared cells of replicon RNA. 2CMC was also effective
against MNV in vitro and in vivo, in which treatment prevented
diarrhea and mortality in a lethal mouse model of infection [6,
10]. The same authors recently demonstrated that treatme nt
with 2CMC reduced shedding and transmission of MNV and
that prophylactic treat ment completely protected against
MNV infection [14]. While these results are highly promising,
2CMC has not yet been approved for treatment of HCV
Figure 1. Organization of the human norovirus genome and the main antiviral targets. The genome is covalently attached at the 5′ end to VPg and is polyadenylated at the 3′
end. RNA structures are present at either end of the genome, which interact with host cell factors (shaded shapes) to achieve replication and translation. Essential host cell
factors represent potential antiviral targets for small-molecule inhibitors. The viral genome is divided into 3 open reading frames (ORFs). ORF1 encodes the viral polyprotein,
which is cotranslationally and posttranslationally cleaved by the viral protease, NS6
pro
, to release mature nonstructural proteins, including the viral RNA–dependent RNA
polymerase (RdR p). Names given in brackets for the nonstructural proteins represent the alternative nom enclature used for murine norovirus. Both NS6
pro
and the RdRp
are key viral protein targets for a number of inhibitors as shown. ORF2 and ORF3 are translated from the subgenomic RNA and encode the major and minor capsid proteins
respectively. The antiviral effects of type I and II interferons (IFNs) are thought in part to be mediated at the level of translation, although the mechanism of action of IFN-λ has
not yet been determined. Abbreviation: 2CMC, 2′-C-methylcytidine.
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infection because of concerns regarding its toxicity profile in pa-
tients, although derivatives of 2CMC are currently under devel-
opment and could prove of potential therapeutic use.
Several non-nucleoside inhibitors have been identified with
activi ty against the HuNoV RdRp in enzymatic assays. These
include suramin, NF203, and PPNDS [10]. Crystallographic
studies have revealed the binding site of each compound on
the RdRp, but they have yet to be tested in the cell culture or
animal models of norovirus infections. Recently, a high-
throughput screen against HuNoV GII.4 RdRp activity identi-
fied several compounds that also in hibi ted the Norwalk virus
replicon and MNV replication in vitro, although at much higher
concentrations. These compounds now provide a sc aff old for
rational design and optimization of specific HuNoV RdRp in-
hibitors [15].
TARGETING THE HOST CELL AS AN ANTIVIR AL
APPROACH
Owing to the high error rates of RNA virus replication, norovi-
rus included, the efficacy of antivirals targeting viral proteins is
often limited by the emergence of drug-resistance mutations.
An alternative strategy that circumvents this problem is to target
host cell proteins that are essential for viral replication, provid-
ing a much higher barrier to the generation of resistant mutants.
A number of host cell factors have been identified that interact
with the viral genome and are required for either its replication
or translation; these include La, PTB, DDX3, PCPB2, and
hnRNPs [6]. RNAi-mediated knockdown of these factors re-
duced MNV viral yields in vitro, demonstrating the potential
of targeting host cell proteins to restrict replication. Modulation
of entire cellular processes has also been shown as an antiviral
approach [16]. WP1130, a small-molecule inhibitor of cellular
deubiquitinases, inhibits replication of MNV and several
other RNA viruses, with this activity indirectly mediated by ac-
tivation of the unfolded protein response (UPR). Inhibition of a
specific cellular deubiquitinase (UPS14) resulted in the activa-
tion of inositol-requiring enzyme, a key mediator of the UPR, in
which endoplasmic reticulum–associated protein degradation is
increased and cellular translation decreased. Small-molecule ac-
tivators of the UPR also reduced MNV replication. Derivatives
of WP1130 have recently been identified that have enhanced
broad antiviral activity, without cellular toxicity, although
they have yet to be evaluated in vivo [16].
LETHAL MUTAGENESIS OF NOROVIRUSES
Lethal mutagenesis has recently emerged as a novel alternative
strategy to classical antiviral approaches. Several nucleoside an-
alogues, such as r ibavirin and favipiravir, display antiviral
Figure 2. Lethal mutagenesis as an antiviral strategy to control norovirus. Top, During multiple rounds of virus infection in host cells, diverse virus populations (known as
quasispecies) are formed as a result of low viral RNA–dependent RNA polymerase replication fidelity. Genetic diversity is represented as different virus particles containing
different shape and color symbols. Some viruses will contain lethal mutations and are naturally non-viable, represented with a cross. Genetic diversity enables RNA viruses to
have rapid adaptability to the environment and the flexibility to escape from immune responses and antiviral compounds. Bottom, Lethal mutagenesis exploits low replication
fidelity to drive RNA viruses to extinction through an excessive accumulation of mutations. A mutagenic compound (black star) interferes with virus replication, leading to larger
mutation frequencies. As a result of increased error rates during lethal mutagenesis, a larger proportion of viruses will contain lethal mutations. Continuous replication in the
presence of an efficient mutagen can result in complete loss of viral infectivity (virus extinction).
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activities associated with induced mutagenesis in vitro [17, 18].
Excessive accumulation of mutations during replication, beyond
a tolerated value known as the error threshold, can lead to virus
extinction, a proce ss known as lethal m utagenesis (Figure 2).
The in vitro evidence has led to several studies that assessed
the clinical use of lethal mutagenesis. Recent reports on HCV
have suggested that the antiviral activity of ribavirin in vivo
may be a ssociated with increased mutagenesis, although this
is still a controversial topic [18].
With the aim of assessing antiviral therapies based on lethal
mutagenesis, we have recently investigated whether ribavirin
and favipiravir are also mutagenic for noroviruses [ 17]. We
found that treatment of MNV-infected mice with favipiravir re-
sulted in more-rapid clearance rates of infectious virus. Viral
populat ions from treated mice showed highe r mutation fre-
quencies and lower replication fitness than viruses isolated
from control mice, suggesting that favipiravir can drive persis-
tent MNV infections to extinction through the accumulation of
debilitating mutations. We also showed that the antiviral activ-
ity of favipiravir is sustained during prolonged treatment peri-
ods (>50 days) without any observed rebound in viral titers. Our
data also sugg ested that favipiravir is more efficient than ribavi-
rin in the control of MNV infections in vivo, possibly because
favipiravir induces more-effective increases in mutat ion fre-
q uency. Ribavirin and favipiravir are purine anal ogues that
can be triphosphorylated within the cell and then incorporated
into nascent viral genomes by the viral RdRp. Their mutagenic
activity resides in their ambiguous pairing behavior, establish-
ing stable base pairing with both uridine and cytidine. This am-
biguity can occur as they are incorporated into the nascent viral
RNA and in the next round of replication acting in the template
strand, resulting altogether in an increase in the number of tran-
sition mutations.
The main advantage of lethal mutagenesis relative to other
antiviral strategies is that it causes a continuous attenuation of
the replicating virus, leading to clearance of infection. In the
absence of the drug, attenuating deleterious mutations may re-
main imprinted in the viral population and in any transmitted
viru s. In addition, it has been demonstrated that significantly
attenuated and defective variants generated during mutagenesis
can interfere with infection by replication-competent viruses,
thereby facilitating viral extinction [18]. These results raise the
possibility of using favipiravir and lethal mutagenesis as an an-
tiviral strategy to treat chronic HuNoV infections in immuno-
com promised individuals. A recent study has indicated that
ribavirin may be effective in some chronically infected patients,
although whether the antiviral activity observed was due to mu-
tagenesis was not examined [19]. Clearly, further studies in this
area are warranted.
Recent studies encourage the development of new combina-
tional approaches involving a mutagenic compound and a clas-
sical inhibitor, to improve the efficacy of therapies based on
lethal mutagenesis [18]. A possible limitation of such cocktails
may be the greater likelihood of resistance to classical inhibitors
as a consequence of the increased mutation frequencies. Al-
ternative combinati onal approaches may involve sequential
administration of inhibitor and mutagen to reduce the possibil-
ity of resistance emerging, a regimen recently shown to be more
efficient than simultaneous administration [18]. Combinations
of mutag ens and inhi bitors targeting critical host factors may
also present a rational approach, with a lower risk of generating
viral resistance.
SUMMARY
In recent years, there has been significant progress in the devel-
opment of small-molecule inhibitors for HuNoV (Table 1),
driven by different approaches and accompanied by increased
understanding of the norovirus life cycle and development of
new systems for studying norovirus replication. Importantly,
however, further studies will be required to bring any of these
potential compounds through clinical trials and into the clinic.
To date, the only reported preliminary clinical trial tested nita-
zoxanide, a compound licensed against certain parasites, which
produced a modest decrease in symptom duration in healthy in-
dividuals but, more imp ortantly, resolved symptoms within a
single chronically infected patient [10]. However , no furth er
use has been reported since 2011. Evaluating inhibitors that
are currently in trials or under development for other viruses
with conserved protein functions, including 2CMC derivatives
and favipiravir, may serve to accelerate treatments for HuNoV
through clinical trials.
Su pplementary Data
Supplementary materials are available at http://jid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Table 1. Summary of Potential Norovirus Antivirals
Compound Target Mechanism of Action
Classical inhibitor
IFN-λ Host cells Induction of antiviral state, specific
mediators unknown [11]
Rupintrivir Viral protease Irreversible inhibitor of active site [12]
2CMC Viral polymerase Nucleoside analogue [16]
Suramin Viral polymerase Nonnucleoside analogue [10]
NF203 Viral polymerase Nonnucleoside analogue [10]
PPNDS Viral polymerase Nonnucleoside analogue [10]
Host cell inhibitor
WP1130 Cellular
deubiquitinases
Indirect activation of the unfolded
protein response [16]
Chemical mutagen
Favipiravir Viral polymerase Lethal mutagenesis [17]
Ribavirin Viral polymerase Lethal mutagenesis [17]
Abbreviations: 2CMC, 2′-C-methylcytidine; IFN-λ, interferon λ.
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Notes
Financial support. This work was supported by the Wellcome Trust
(reference WT097997MA; senior fellowship to I. G.).
Potential conflicts of interest. All authors: No reported co nflicts. All
authors have submitted the ICMJE Form for Disclosure of Potential Con-
flicts of Interest. Conflicts that the editors consider relevant to the content
of the manuscript have been disclosed.
References
1. Payne DC, Vinje J, Szilagyi PG, et al. Norovirus and medically attended gastroen-
teritis in U.S. children. N Engl J Med 2013; 368:1121–30.
2. Harris JP, Adams NL, Lopman BA, Allen DJ, Adak GK. The development of web-
based surveillance provides new insights into the burden of norovirus outbreaks in
hospitals in England. Epidemiol Infect 2014; 142:1590–8.
3. Ronchetti AM, H enry B, Amber t-Balay K, et al. Norovirus-related chronic
diarrhea in a patient treated with alemtuzumab for chronic lymphocytic leukemia.
BMC Infect Dis 2014; 14:239.
4. Woodward JM, Gkrania-Klotsas E, Cordero-Ng AY, et al. The role of ch ronic
norovirus infection in the enteropathy associated with common variable immuno-
deficiency. Am J Gastroenterol 2015; 110:320–7.
5. Patel MM, Widdowson M-A, Glass RI, Akazawa K, Vinjé J, Parashar UD. System-
atic literature review of role of noroviruses in sporadic gastroenteritis. Emerg Infect
Dis 2008; 14:1224–31.
6. Arias A, Emmott E, Vashist S, Goodfellow I. Progress towards the prevention and
treatment of norovirus infections. Future Microbiol 2013; 8:1475– 87.
7. Jones MK, Watanabe M, Zhu S, et al. Enteric bacteria promote human and mouse
norovirus infection of B cells. Science 2014; 346:755–9.
8. Taube S, Kolawole AO, Hohne M, et al. A mouse model for human norovirus.
MBio 2013; 4:00450-13.
9. Katayama K, Murakami K, Sharp TM, et al. Plasmid-based human norovirus
reverse genetics system produces reporter-tagged progeny virus containing infec-
tious genomic RNA. Proc Natl Acad Sci U S A 2014; 111:5.
10. Kaufman SS, Green KY, Korba BE. Treatment of norovir us infections: moving
antivirals from the bench to the bedside. Antiviral Res 2014; 105:80–91.
11. Nice TJ, Baldridge MT, McCune BT, et al. Interferon lambda cures persi stent
murine norovirus infection in the absence of adaptive immunity. Science 2014;
27:1258100.
12. Rocha-Pereira J, Nascimento MS, Ma Q, Hilgenfeld R, Neyts J, Jochmans D. The
enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus ac-
tivity and clears cells from the norovirus replicon. Antimicrob Agents Chemother
2014; 58:4675–81.
13. Ferrer-Orta C, Arias A, Escarmís C, Verdaguer N. A comparison of viral RNA-de-
pendent RNA polymerases. Curr Opin Struct Biol 2006; 16:27–34.
14. Rocha-Pereira J, Jochmans D, Neyts J. Prophylactic treatment with the nucleoside
analogue 2′-C-methylcytidine completely prevents transmission of norovirus.
J Antimicrob Chemother 2015; 70:190–7.
15. Eltahla AA, Lim KL, Eden JS, Kelly AG, Mackenzie JM, White PA. Nonnucleoside
inhibitors of norovirus RNA polymerase: scaffolds for rational drug design. Anti-
microb Agents Chemother 2014; 58:3115–23.
16. Gonzalez-Hernandez MJ, Pal A, Gyan KE, et al. Chemical derivatives of a small
molecule deubiquitinase inhibitor have antiviral activity against several RNA vi-
ruses. PLoS One 2014; 9:e94491.
17. Arias A, Thorne L, Goodfellow I. Favipiravir elicits antiviral mutagenesis during
virus replication in vivo. Elife 2014; 3:e03679.
18. Domingo E, Sheldon J, Perales C. Viral quasispecies evolution. Microbiol Mol Biol
Rev 2012; 76:159–216.
19. Woodward JM, Gkrania-Klotsas E, Cordero -Ng AY, et al. The role of chro nic
norovirus infection in the enteropathy associated with common variable immuno-
deficiency. Am J Gastroenterol 2015; 110:320–7.
Advances Toward Norovirus Antivirals
•
JID 2016:213 (Suppl 1)
•
S31
by guest on January 14, 2016http://jid.oxfordjournals.org/Downloaded from