The Role of Zinc in Antiviral Immunity
1,2Stephanie Obeid,3Chantelle Ahlenstiel,3andGoloAhlenstiel
1Blacktown Medical School, Western Sydney University, Blacktown, New South Wales, Australia; 2Storr Liver Centre, The Westmead Institute for Medical
Research, The University of Sydney and Westmead Hospital, Westmead, New South Wales, Australia; and 3The Kirby Institute, University of New South Wales,
Sydney, New South Wales, Australia
Zinc is an essential trace element that is crucial for growth, development, and the maintenance of immune function. Its inﬂuence reaches all organs
and cell types, representing an integral component of approximately 10% of the human proteome, and encompassing hundreds of key enzymes
and transcription factors. Zinc deﬁciency is strikingly common, aﬀecting up to a quarter of the population in developing countries, but also aﬀecting
distinct populations in the developed world as a result of lifestyle, age, and disease-mediated factors. Consequently, zinc status is a critical factor that
can inﬂuence antiviral immunity, particularly as zinc-deﬁcient populations are often most at risk of acquiring viral infections such as HIV or hepatitis
C virus. This review summarizes current basic science and clinical evidence examining zinc as a direct antiviral, as well as a stimulant of antiviral
immunity. An abundance of evidence has accumulated over the past 50 y to demonstrate the antiviral activity of zinc against a variety of viruses,
and via numerous mechanisms. The therapeutic use of zinc for viral infections such as herpes simplex virus and the common cold has stemmed
from these ﬁndings; however, there remains much to be learned regarding the antiviral mechanisms and clinical beneﬁt of zinc supplementation
as a preventative and therapeutic treatment for viral infections. Adv Nutr 2019;0:1–15.
Keywords: zinc, virus, metallothionein, antiviral, immunity, zinc deﬁciency, zinc supplementation
Zinc deciency was rst recognized by Prasad et al. >50 y
ago in a malnourished group of individuals presenting
with hepatosplenomegaly, dwarsm, hypogonadism, and an
elevated risk of infection (1). Unbeknownst to Dr. Prasad and
his colleagues at the time, their discovery would highlight
the importance of zinc as an integral component of human
physiology, and inspire decades of zinc research. It is now
understood that zinc is the second-most abundant trace
ponent of protein structure and function. Importantly, zinc
is a structural constituent of ∼750 zinc-nger transcription
factors (2) enabling gene transcription, and is a catalytic
component of approximately 2000 enzymes, encompassing
all 6 classes (hydrolase, transferase, oxido-reductase, ligase,
lyase, and isomerase) (3). Hence, zinc is biologically essential
Supported by Sylvia and Charles Viertel Charitable Foundation Investigatorship (VTL2015C022).
Author disclosures: SAR, SO, CA, and GA, no conicts of interest.
Address correspondence to SAR (e-mail: email@example.com).
Abbreviations used: EV, epidermodysplasia verruciformis; HCMV, human cytomegalovirus; HCV,
hepatitis C virus; HPV, human papilloma virus; HSV,herpes simplex virus; IRF, IFN regulatory
factor; ISG, interferon stimulated gene; MT, metallothionein isoforms; MTF1, metal-responsive
transcription factor; PRR, pattern recognition receptor; RdRp, RNA-dependent RNA polymerase;
RT, reverse transcriptase; SOCS, suppressors of cytokine signaling; TLR, Toll-like receptor; ZIP,
Zrt- and Irt-like proteins.
well as DNA synthesis and RNA transcription (4).
The global prevalence of zinc deciency is estimated
to range from ∼17% to 20% (5,6), with the vast ma-
jority occurring in developing countries of Africa and
Asia. Although signicantly less common in high-income
nations, zinc deciency occurs most frequently in the elderly,
vegans/vegetarians, and individuals with chronic disease
such as liver cirrhosis (7)orinammatoryboweldisease
(8). Importantly, zinc deciency results in a compromised
immune system, as evidenced by thymic atrophy, lymphope-
nia, and defective lymphocyte responses in animal studies
(9). These data underscore the importance of zinc nutrition,
particularly in underdeveloped countries where the risk of
infection is heightened because of poor sanitation, public
health, and vaccination strategies (5).
This review focuses on the role of zinc as an essential
micronutrient that is required to mount an eective antiviral
response. Although zinc possesses direct antiviral properties
(e.g. inuenza), it is also critical in generating both innate
and acquired (humoral) antiviral responses. To complicate
matters, zinc is an integral component of many viral enzymes,
proteases, and polymerases, highlighting the importance of
regulating cellular and systemic zinc distribution to inhibit
viral replication and dissemination.
American Society for Nutrition 2019. All rights reserved. Adv Nutr 2019;0:1–15; doi: https://doi.org/10.1093/advances/nmz013. 1
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Current Status of Knowledge
Zinc homeostasis and viral infection
Systemic and intracellular zinc are tightly regulated, such
that free zinc ions (Zn2+) represent a minimal fraction of
total cellular zinc (∼0.0001%) (10–12). The vast majority
of zinc remains bound to zinc-binding proteins such as
serum albumin or intracellular metallothionein proteins,
where it can be transferred to zinc-binding enzymes and
transcription factors as necessary. Zinc transport is princi-
pally mediated by 2 groups of proteins: the ZnT [solute-
linked carrier 30 (SLC30A)] family, which is responsible
and the ZIP [Zrt- and Irt-like proteins (SLC39A)] family
of proteins, which performs the opposite role, transporting
zinc into the cytoplasm from extracellular sources or cellular
organelles (13). The >30 human proteins responsible for zinc
toxic in the case of dietary excess, nor limited in the case
of dietary insuciency. Of course, this balance cannot be
maintained indenitely, and may result in zinc-induced
copper deciency if consumed in excess (14), and severe zinc
deciency if it is lacking in the diet (1).
Sequestration and toxic accumulation of metals are well-
documented antibacterial immune responses. Calprotectin
is a prime example, binding and sequestering extracellular
calcium and zinc, thus preventing bacterial and fungal
overgrowth (15). Conversely, toxic endosomal zinc accu-
mulation can inhibit intracellular Mycobacterium growth in
macrophages (16). Unfortunately, these mechanisms are not
well described in the case of viral infections, perhaps because
of a lack of ecacy. Calprotectin, for example, has no proven
antiviral role, nor is it signicantly upregulated in response
to viral gastroenteritis (17). This absence of a zinc-mediated
antiviral response may reect the “parasitic” nature of viral
infection, hijacking host machinery to self-replicate. Changes
in intracellular zinc concentrations necessary to inhibit viral
replication may also prove toxic to eukaryotic cells for the
Although antiviral modulation of zinc homeostasis in
humans remains unproven, papilloma viruses have evolved
mechanisms to alter zinc homeostasis to favor viral replica-
tion and persistence (18). The human papilloma virus (HPV)
E5 protein can interact with the zinc transporter ZnT-1 in
complex with EVER2, thus stimulating nuclear accumulation
of zinc (19). The ZnT-1:EVER2 complex responsible for zinc
export from the nucleus is inhibited by HPV E5, subsequently
increasing both nuclear zinc and the activation of AP1 (20),
a transcription factor required for HPV genome expression.
Interestingly, homozygous mutations in either EVER1 or
EVER2 result in a rare condition termed epidermodysplasia
HPV strains 5 and 8, which signicantly increases the risk of
developing nonmelanoma skin cancers. HPV strains 5 and 8
lack expression of the E5 protein, which may explain 1)their
limited replication in the normal population because of their
inability to control zinc homeostasis, and 2)thesusceptibility
of EV patients to strains 5 and 8 from the loss of EVER protein
function, favoring HPV replication. Interestingly, HPV E5
genes have co-evolved with the major HPV oncogenes, E6
and E7, and indicate the potential involvement of E5 in
carcinogenesis (21,22). Clinical trials using both oral and
topical zinc have proven eective for the treatment of viral
Metallothioneins, zinc homeostasis, and antiviral activity.
Metallothioneins are small, cysteine-rich proteins capable
of binding divalent cations such as zinc and copper. As
vessels for much of the labile intracellular zinc pool,
metallothioneins possess numerous functions through their
ability to bind and release metals from their thiol groups.
These include storage and transfer of zinc ions and heavy
metal detoxication, as well as involvement in oxidative
stress, apoptosis, and immune responses (23). Humans
express 4 metallothionein isoforms (MT1–4), including the
ubiquitously expressed MT1 and MT2 genes (MT1A, B,
E, F, G, H, I, J, L, M, X, MT2A), as well as MT3 and
MT4 whose expression is limited, and function remains
poorly understood (24). Importantly, MT1 and 2 gene
expression is extremely responsive to zinc, and therefore
serves as an ideal indicator of an individual’s zinc status (25).
Upon taking a zinc supplement, for example, an increase
in protein-bound zinc in the bloodstream is internalized
by cells in various tissues and organs through the ZIP
transporters. In response to increased intracellular zinc,
the metal-responsive transcription factor (MTF1) becomes
active, and binds the metal responsive element in metal-
lothionein gene promoters to upregulate their transcription
(26). Although there are additional stimuli that inuence
metallothionein expression, this primarily occurs in a zinc-
dependent fashion. Oxidative stress, for example, induces
zinc release from metallothioneins as a mechanism to reduce
reactive oxygen species generated by mitochondrial dysfunc-
tion or viral infection (26). Zinc released from metalloth-
ioneins binds MTF1 to stimulate additional metallothionein
It should be noted that metallothioneins, although highly
responsive to zinc, have long been classied as interferon
stimulated genes (ISGs) (27). IFNs are immunostimulatory
cytokines secreted from infected cells and nearby immune
cells that induce the expression of hundreds of antiviral
genes. They possess diverse roles including chemoattraction,
immune cell activation, and direct antiviral activity. In
metallothionein induction. Most ISGs possess binding sites
for STAT- or IFN regulatory factor (IRF) transcription factor-
mediated expression, as is the case for MT1X and MT2A (28,
29). Other metallothioneins such as MT1F and MT1G do not
possess known IFN regulatory regions in their promoters,
but are instead more sensitive to zinc (28). IFNs stimulate
an inux of zinc into the target cell, as is the case with some
inammatory cytokines such as IL-6, which in turn drives
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Because metallothioneins possess such a diverse func-
tional repertoire, their specic roles during viral infection
remain undened. However, both in vitro and in vivo
studies have made it abundantly clear that metallothioneins
are induced by viruses. The mechanisms often remain
undened; however, metallothionein expression has been
attributed to zinc inux or redistribution (19,28), by
viral means, cytokine exposure, or oxidative stress (30).
Metallothionein upregulation has been observed in response
to measles virus (31), inuenza (31,32), HIV (33), hepatitis
Cvirus(HCV)(34), and coxsackie virus (35), among others.
In the case of HIV, zinc appears to be the key driver of
metallothionein expression to favor viral persistence. HIV-
infected monocytes demonstrate a signicant increase in
both MT1 gene expression as well as intracellular zinc (33).
Elevated intracellular zinc increases monocyte resistance to
apoptosis via inhibition of caspase 3 activation [as has been
reported previously (36)], thus providing a reservoir for HIV
replication. The role of metallothioneins remains unclear in
this study; however, they have been described as negative
regulators of apoptosis, albeit not through direct caspase
3inhibition(37). Zinc and metallothioneins also facilitate
human cytomegalovirus (HCMV) replication by activating
the immediate-early HCMV promoter (38,39). Kanekiyo et
al. demonstrated that both zinc and metallothionein overex-
pression increased NF-κB binding in the HCMV promoter.
Because no complex was detected between metallothionein
and NF-κB, it was suggested that metallothioneins served
as a zinc donor necessary for NF-κB binding. In addition,
as NF-κB transcription factors are known potent activators
of HIV and HSV replication, and several other viruses
(40), metallothioneins may be proviral. Zinc has also been
reported to inhibit NF-κB in numerous studies (41–43).
Despite these contrasting data, Kim et al. have bridged these
inconsistencies, demonstrating that MT2A can serve as a sink
for excess zinc (44), thus limiting its proximity to NF-κBand
favoring NF-κB-mediated transcription.
In the case of HCV infection, metallothioneins possess
an antiviral role. Using a pan-metallothionein siRNA to
knockdown all MT1 and 2 genes, we demonstrated both an
increase in HCV replication and a decrease in intracellular
zinc content in vitro (34). Interestingly, although ZnSO4can
reduce HCV replication, this eect was ablated when metal-
lothionein genes were knocked down. These data suggest that
metallothioneins are either 1) directly antiviral, potentially
by sequestering zinc away from viral metalloproteins such
as HCV NS5A (45), or 2) indirectly antiviral by acting as
zinc chaperones and facilitating antiviral signaling. Further,
metallothioneins possess antiviral properties against other
viruses as well, as demonstrated in an antiviral screen
of 380 human ISGs performed by Schoggins et al. (46).
Overexpression of multiple members of the MT1 family
inhibited replication of aviviruses including yellow fever
virus and HCV, as well as the alphavirus Venezuelan equine
encephalitis virus. This eect was not observed in West
Nile virus, and Chikungunya virus. These data indicate that
metallothioneins, like many ISGs, are selectively antiviral,
perhaps reecting specic viral zinc requirements during
replication. This is particularly evident for HIV, which
demonstrated an increase in viral replication as a result of
metallothionein overexpression in the Schoggins et al. ISG
screen (46), validating previous works (33).
Zinc as an antiviral: bench to bedside and back again
antiviral agent in vitro. Unfortunately, zinc concentrations
used to assess antiviral activity often far exceed physiological
concentrations. Human plasma zinc, for example, ranges
from approximately 10 to 18 μM(47), whereas antiviral
concentrations of zinc can reach into mM concentrations
(48). Intracellular zinc concentrations range from 10s to 100s
of μM, but are signicantly buered by zinc-binding proteins
such as metallothioneins, rendering free zinc concentrations
at picomolar to low nanomolar concentrations (49,50). The
antiviral properties of zinc are certainly virus-specic, but
it would appear that zinc ion availability plays a signicant
role in the antiviral ecacy of zinc (51). Here we describe
the role of zinc as a virus-specic antiviral: both in vitro
mechanistic studies, as well as human-based clinical trials
using zinc supplementation. In vitro and in vivo studies are
summarized in Tab l es 1 and 2,respectively.
The eect of zinc on HSV-1 and -2 has been studied for >40 y,
with in vitro studies suggesting that zinc plays an inhibitory
role on almost every aspect of the viral life cycle: viral
polymerase function (52),proteinproductionandprocessing
(53), and free virus inactivation (48,54). Although these
studies were performed >20 y ago, a more recent study using
the zinc ionophore pyrithione demonstrated a reduction
in HSV replication from reduced NF-κBactivationby
interfering with the protein ubiquitination pathway (41).
Unfortunately, no recent experimental data can demonstrate
with any certainty the mechanism by which zinc inhibits
HSV infection. Nonetheless, in vivo studies in mice and
humans have shown a signicant reduction of infection and
disease burden. Mouse studies performing intravaginal zinc
inoculation in liquid (55)orgel(56)formbothresulted
in signicant reductions in HSV-2 infection. Several topical
zinc application studies have been performed in humans,
which demonstrated a signicantly reduced recurrence and
duration of infection (outbreak) (57–58). The ecacy of
topical application, together with in vitro results (48,54),
suggest that free zinc may indeed coat HSV virions, thus
preventing infection. Further research into this molecular
mechanism is warranted.
Apart from HCMV mentioned above, the eect of zinc
on other members of the Herpesviridae family remains
unknown because of a lack of clinical data. Mechanistically,
zinc ions have been shown to inhibit Varicella-Zoster virus by
inactivating free virus in vitro (59). Both HSV and Varicella-
Zoster virus belong to the Alphaherpesvirinae subfamily,
reecting their genetic relatedness, and similar mechanism
Zinc as an antiviral 3
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TABLE 1 In vitro studies assessing the antiviral ecacy of zinc1
Virus Antiviral eﬀect Zinc Eﬀective dose Reference
Coronavirus Inhibition of RdRp template binding and elongation PT +Zn(OAc)22–320 μMPT+2–500 μMZn (60)
Encephalomyocarditis virus Inhibition of viral polyprotein cleavage ZnCl20.4–1.5 mM (61)
Inhibition of viral polyprotein cleavage ZnCl20.1–1 mM (62)
Inhibition of viral polyprotein tertiary structure PT, HK 5–20 μM PT, 60–125 μMHK (63)
Inhibition of viral polyprotein tertiary structure PDTC 15–125 μMPDTC (63)
Foot and mouth disease virus Inhibition of viral polyprotein cleavage ZnCl2,Zn(OAc)
20.1–2 mM (64)
Inhibition of viral RNA and procapsid synthesis ZnCl210–150 μM(65)
Hepatitis C virus Inhibition of RNA polymerase ZnCl260–300 μM(66)
Inhibition of viral replication ZnCl2,ZnSO
Metallothionein-dependent inhibition of viral replication ZnSO450 μM(34)
Herpes simplex virus Viral protein synthesis ZnSO4N/A (53)
Inhibition of viral DNA polymerase Zn(OAc)20.1–2 mM (52)
Free virus inactivation ZnSO40.1–6 mM (48)
Free virus inactivation Zn(Glu)2, Zn(Lac)21–50 mM (54)
Inhibition of protein ubiquitination and NF-κB activity PT 1.2–18.9 mM (41)
Human immunodeﬁciency virus HIV protease inhibition Not listed 0.2–2 mM (68)
Inhibition of viral transcription and particle production ZnCl270–700 μM(69)
Inhibition of reverse transcriptase ZnCl225–800 μM(70)
Human papilloma virus Stimulates proviral transcription factor activity, reversed
N/A N/A (19)
Inhibition of viral protein E6 and E7 synthesis stimulating
CIZAR 500–750 μM(71)
Respiratory syncytial virus Reduction in viral titer and plaque count ZnCl2,Zn(OAc)
2, Zn(Lac)20.01–10 mM (72)
Rhinovirus Inhibition of viral polyprotein cleavage ZnCl2100–800 μM(73,74)
Inhibition of viral polyprotein cleavage ZnCl20.1–1.2 mM (61)
Inhibition of viral polymerase not listed >0.6 μM(75)
Inhibition of viral polyprotein processing PT, HK 5–20 μM PT, 60–125 μMHK (63)
Inhibition of viral polyprotein processing PDTC 15–125 μMPDTC (63)
Semliki Forest virus Inhibition of endosomal membrane fusion ZnCl225–100 μM(76)
Inhibition of endosomal membrane fusion ZnCl22mM (77)
Sindbis virus Inhibition of viral particle production and polyprotein
ZnCl20.1–1.8 mM (78)
Transmissible gastroenteritis virus Inhibition of viral RNA and protein synthesis ZnCl2,ZnSO
Vaccinia virus Inhibition of RNA synthesis and viral yield ZnSO4100–300 μM(80)
Inhibition of viral particle production and polyprotein
Inhibition of viral topoisomerase Not listed 2.5 mM (82)
Varicella-zoster virus Free virus inactivation Zn(Pic)2,Zn(Asp)
1CIZAR, zinc citrate compound; HK, hinokitiol; N/A, not applicable; PDTC, pyrrolidine-dithiocarbamate; PT, pyrithione; RdRp, RNA-dependent RNA polymerate; Zn(Asp)2, zinc aspartate; ZnCl2, zinc chloride; Zn(Glu)2, zinc gluconate; Zn(Lac)2,zinc
lactate; Zn(OAc)2, zinc acetate; Zn(Pic)2, zinc picolinate; ZnSO4, zinc sulfate.
TABLE 2 Human clinical studies using zinc as an antiviral therapy 1
Viral infection/condition Antiviral/therapeutic eﬀect Eﬀective dose Treatment Reference
Torque teno virus Reduced viral load following stem cell transplant 600 mg ZnSO4/d Oral (83)
Herpes simplex Reduced duration and severity of outbreak ZnO/glycine cream (0.3% ionic Zn) Topical (57)
Reduction in outbreak recurrence 0.025% ZnSO4solution Topical (84)
Reduction in outbreak recurrence 1–4% ZnSO4solution Topical (58)
Experimental rhinovirus Reduced duration of illness with Zn(Glu)2only Zn(Glu)2(13.3 mg) or Zn(OAc)2(5/11.5 mg) lozenges, every 2–3 h/d Lozenge (85)
Common cold Reduced symptom severity, frequency, and duration ZGG lozenges containing 23 mg Zn, every 2 h/d Lozenge (86)
Reduced symptom severity, frequency, and duration ZGG lozenges containing 24 mg Zn, every 2–3 h/d (Max 8) Lozenge (87)
Reduced duration of symptoms ZGG lozenges containing 13 mg Zn, every 2 h/d Lozenge (88)
Reduced symptom severity and duration Zn(OAc)2lozenges each containing 9 mg Zn, every 2 h/d Lozenge (89)
Reduced symptom severity and duration Zn(OAc)2lozenges each containing 13 mg Zn, every 2–3 h/d Lozenge (90)
No eﬀect on duration or severity Zn(Glu)2(13.3 mg) or Zn(OAc)2(5/11.5 mg) lozenges, every 2–3 h/d Lozenge (85)
Reduced symptom severity and duration Zn(OAc)2lozenges each containing 13 mg Zn, every 2–3 h/d Lozenge (91)
Viral warts Improved clearance of warts after 1–2 mo 10 mg/ kg ZnSO4to a maximum dose of 600 mg/d Oral (92)
Clearance of warts based on concentration of zinc used 3 ×5 or 10% ZnSO4/d Topical (93)
Improved clearance of warts after 1–2 mo 10 mg/ kg ZnSO4to a maximum dose of 600 mg/d Oral (94)
No beneﬁt 10 mg/ kg ZnSO4/d Oral (95)
Resolution of 88% of lesions after 6 wk/3 sessions Up to 3 intralesional injections with 2% ZnSO4Injection (96)
Laryngeal papillomatosis Resolution of papillomatosis (2 case studies) 10 mg/ kg ZnSO4/d Oral (97)
HIV Reduced infection, increased CD4 T cell count 200 mg/d ZnSO4/d Oral (98)
Increased CD4 T cell count 45 mg Zn(Glu)2every8hfor15d,then15mgfor15d Oral (99)
Reduced incidence of diarrhea 10 mg elemental zinc as ZnSO4/d Oral (100)
No beneﬁt 25 mg/d ZnSO4/d Oral (101)
Chronic hepatitis C virus Enhanced response to IFN treatment 2 ×75 mg polaprezinc/d Oral (102)
No beneﬁt to IFN treatment response 5 ×78 mg Zn(Glu)2/d Oral (103)
Reduced serum AST, ALT, and ferritin 3 ×75 mg polaprezinc/d Oral (104)
Reduced serum ALT and Th2 cells (%) 2 ×75 mg polaprezinc/d Oral (105)
Reduced incidence of HCC (albumin-dependent) 2 ×150 mg polaprezinc/d Oral (106)
1ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCC, hepatocellular carcinoma; ZGG, zinc gluconate/glycine; Zn(Glu)2, zinc gluconate; ZnO, zinc oxide; Zn(OAc)2,zincacetate;ZnSO
4, zinc sulfate.
Zinc as an antiviral 5
It was clear as early as 1974 that zinc possessed an inhibitory
eect on picornovirus polyprotein processing (73). Before
1980, zinc inhibition of picornovirus proteases from human
rhinovirus isolates (73,74), encephalomyocarditis virus (62),
poliovirus (61), and foot and mouth disease virus (64,65)
had all been demonstrated. More recent studies using zinc
ionophores have illustrated that zinc interferes with the
autocatalytic processing of the viral protease 3CDpro into
3Cpro in the picornavirus coxsackievirus B3, thus inhibiting
processing of the viral polyprotein (107). However, this was
not the case for encephalomyocarditis virus, where zinc
appeared to inhibit the tertiary structure within the viral
polyprotein (107). Together, these data suggest that zinc may
interfere with proteolytic processing of the viral polyprotein
because of misfolding, or through direct actions on the viral
Clinical studies using zinc supplementation are primarily
limited to rhinovirus infection, and are often grouped
with other “common cold” viruses such as inuenza and
coronaviruses. The majority of studies use zinc lozenges with
various zinc formulations and concentrations, possibly ex-
plaining the large variability in results [extensively reviewed
in (108)and(109)]. Importantly, the amount of ionic zinc
present at the site of infection (oral and nasal mucosa) is
highly correlated to the study outcome (51,108), and is
dependent on the zinc formulation. At a physiological pH
and 37◦C, zinc gluconate for example, releases high amounts
of ionic zinc, whereas zinc aspartate releases none (108).
Upon examining only the relevant studies where high doses
of ionic zinc were used, a clear reduction in cold duration
of 42% was calculated (109). Whether this was caused by
viral inhibition, improved local immune response, or an
amelioration of symptoms remains uncertain.
Other respiratory tract infections: inuenza, coronavirus,
Few studies have examined the antiviral eects of zinc on
other respiratory viruses. In vitro replication of inuenza
(PR/8/34) is signicantly inhibited by the addition of the
zinc ionophore pyrrolidine dithiocarbamate (110), perhaps
through inhibition of the RNA-dependent RNA polymerase
(RdRp), as had been suggested 30 y earlier (111). In similar
fashion, severe acute respiratory syndrome (SARS) coron-
avirus RdRp template binding and elongation was inhibited
by zinc in Vero-E6 cells (60). Moreover, zinc salts were shown
to inhibit respiratory syncytial virus, even while zinc was
removed (72). The authors suggest that this indicates an
inhibitory mechanism similar to HSV by preventing viral
membrane fusion; however, no measures were taken to assess
changes in intracellular zinc content, nor inhibition of other
aspects of the viral life cycle.
Flaviviridae: a focus on HCV.
Flaviviruses represent a number of insect-borne viruses
including dengue and West Nile virus, as well as the
hepatotrophic virus, HCV. The eect of zinc on insect-borne
aviviruses is scarce; however, in vitro studies by our group
(34)andothers(67) have demonstrated that zinc salts can
reduce HCV replication (∼50% at 100 μMZnSO
by inhibiting the HCV RdRp, as shown in E. coli [half
maximal inhibitory concentration (IC50) ∼60 μM] (66).
Although this is a potential mechanism, it has not been
examined in eukaryotic cells in which zinc homeostasis is
If left untreated, HCV becomes a chronic hepatic infection
in around two-thirds of individuals (112), resulting in a
signicant reduction in plasma zinc (113). Consequently,
zinc supplementation in HCV studies have focused on
improved patient outcomes, particularly decreased liver
inammation, and enhanced response to antiviral treatment.
Supplementation with 150 mg/d polaprezinc (a bioavailable
of hepatic inammation alanine aminotransferase and aspar-
tate aminotransferase alone (105),andincombinationwith
the antiviral treatment IFN-α(106). Moreover, polaprezinc
signicantly improved the rate of viral clearance, particularly
in patients with lower viral loads at baseline (102). The
mechanisms underlying these observations remain uncer-
tain; however, are likely a combination of direct antiviral
eects and strengthening of the antiviral response. Zinc
supplementation and the antiviral response is reviewed
Togav i ri d a e.
Like aviviruses, togaviruses primarily consist of arthropod-
borne viruses such as Semliki Forest virus, Western equine
encephalitis virus, and Chikungunya virus. Viral infection
occurs by receptor-mediated endocytosis, followed by fusion
of virus and endosomal membranes, and particle release
into the cytoplasm (114). Using liposome (76), red blood
cell (115), and BHK-21 (77) cell model systems, zinc has
been shown to eciently inhibit membrane fusion of Semliki
membrane fusion by binding to a specic histidine residue
revealed on the viral E1 protein at low endosomal pH (77).
Notably, concentrated zinc is present in vesicular zincosomes
that are thought to serve as intracellular zinc storage vesicles
inhibit intracellular Mycobacterium spp., zincosome fusion to
viral endosomes may inhibit key aspects of the viral life cycle
such as togavirus membrane fusion.
Retroviruses are named after their ability to transcribe RNA
into DNA using their unique reverse transcriptase (RT),
consequently allowing integration of retroviral DNA into
the host genome. The integrated provirus can then establish
a latent infection for the life of the host and is a major
barrier to virus cure strategies, particularly for HIV-1 (117).
Similar to viral RdRps, zinc has also been identied as
an inhibitor of retrovirus RTs (118,119). Fenstermacher
and DeStefano demonstrated in 2011 that Zn2+cations
2+ions from HIV-1 RT, promoting the
formation of an excessively stable, but incredibly slow and
inecient replication complex (70). Zinc was also shown
to inhibit the HIV-1 protease in 1991 (68), and to inhibit
viral transcription in 1999 (69), but has received little
attention since, with the exception of molecular simulation
experiments that identied the zinc-binding sites at the
catalytic aspartate-25 residue (120). As stated above, HIV can
also stimulate zinc inux into monocytes (33), which may
appear contradictory based on its antiretroviral properties.
Latently infected monocytes and macrophages, however, can
act as viral reservoirs for HIV (121), and could therefore
benet from zinc-mediated inhibition of cell death. In fact,
unlike the majority of CD4+T cells, low levels of replication
in macrophages do not result in cell death (122), making
them a viable reservoir, in addition to long-lived resting
CD4+T cells, for viral recrudescence after cessation of
Zinc deciency is common in HIV-infected individuals,
where it is associated with inammation (123), immunologi-
cal failure (124), and death (125). A recent Cochrane Review
examined the role of micronutrient supplementation in
people living with HIV (126). Although a number of studies
demonstrated benecial eects of zinc supplementation,
the majority were underpowered. The authors concluded
that zinc supplementation probably increases blood zinc
concentration (moderate certainty), and may increase CD4+
counts (low certainty).
Unlike zinc supplements, prophylactic zinc gels have
shown a substantial benet to limit HIV infection in vivo.
Complete protection against vaginal SHIV-RT (a simian
HIV virus expressing the human RT) infection in macaques
was obtained by pretreating animals with an antiviral gel
containing 14 mM zinc acetate and 50 μM MIV-150, a reverse
transcriptase inhibitor (127). When used alone, zinc acetate is
a potent antiviral, providing 66% protection against SHIV-RT
vaginal infection (56)andanEC50<100 μMinperipheral
blood mononuclear cells against a range of HIV strains
(128). Importantly, zinc treatment did not aect viral titers
in macaques that became infected, nor did it result in zinc
resistant HIV mutants with conserved pol (RT) mutations.
These data suggest that zinc may not interfere with the
HIV RT, but instead inactivate free virus or prevent viral
attachment/penetration as reported for HSV (48,54).
HPVs are oncogenic viruses that infect basal epithelial
cells, where they stimulate proliferation resulting in warts.
Although cutaneous warts are usually self-limiting and
harmless, mucosal strains of HPV (e.g. high risk HPV-16
and -18) are a primary cause of cervical cancers (129).
HPV oncoproteins E6 and E7 in particular, are signicant
drivers of cell proliferation and resistance to cell death by
stimulating the degradation of tumor suppressor p53 and
pRb, respectively [reviewed in (130)]. Although nuclear zinc
appears to enhance HPV replication (see Zinc homeostasis
and viral infection), exogenous zinc treatment (CIZAR, zinc
chloride and citric acid anhydrous) can eectively inhibit
production of viral oncogenic proteins E6 and E7 (71). The
inhibition of E6 and E7 by zinc results in apoptosis of
cervical carcinoma cells, as they regain the function of tumor
suppressors p53 and pRb (71). The mechanism by which zinc
downregulates E6 and E7 expression is unknown, but may be
preceded by a zinc-driven blockade in another component of
the viral life cycle.
It would appear that both topical and oral zinc supple-
mentation strategies have proven tremendously eective for
cutaneous and genital warts. Unfortunately, the vast majority
of studies are either underpowered, lacking suitable controls,
or single case studies. Nonetheless, a recent systematic review
concluded that zinc supplementation was the most eective
systemic treatment for cutaneous warts, when compared to
other available options (131). It should be noted, however,
that individuals with persistent viral warts are often zinc-
decient or have lower concentrations than their healthy
counterparts (132). In fact, studies demonstrating the most
signicant responses to zinc treatment had engaged patients
that were primarily zinc-decient (>70 μg/dL) (92,94).
Nonetheless, 78% (94) and 100% (92)ofpatientsshowed
clearance of lesions in response to oral zinc sulfate (10 mg/kg
up to 600 mg/d) compared to 13% and 0% of the placebo
group, respectively. Topical zinc formulations have also
proved ecacious for treatment of viral warts. A small study
using a 4-wk topical 10% zinc sulfate regimen for plane warts
demonstrated an 86% response rate (6/7), compared to a 10%
response rate (1/10) in the control group (93).
Recent work suggests that treatment of vaginal HPV
infections with topical zinc formulations may benet the
A recent pilot study demonstrated that intravaginal infusion
of 500 μM zinc citrate in women diagnosed with high-risk
HPV resulted in a 64% clearance rate, compared to 15%
in the control group (133). Additional studies in mice have
demonstrated that MZC, a formulation containing MIV-50,
zinc acetate, and carrageenan, eciently inhibited vaginal
and anorectal HPV-16 pseudoviral particle infection (134).
In summary, it is evident that zinc possesses antiviral
properties against a number of viral species. Although
mechanistic studies are lacking, zinc appears to inhibit viral
protease and polymerase enzymatic processes, as well as
physical processes such as virus attachment, infection, and
uncoating (Figure 1). Unfortunately, these mechanisms have
not been well scrutinized in clinical studies, where zinc
may provide inexpensive and eective adjunct treatments for
many viral infections.
The role of zinc in antiviral immune signaling
Ionic zinc possesses unique and distinct antiviral properties
against a number of human viruses; however, the antiviral
immune response led by IFNs is invariably required to clear
infections. Zinc has been shown to contribute to a number
Zinc as an antiviral 7
FIGURE 1 The diverse stages of viral replication cycles that are inhibited by zinc. In vitro studies have demonstrated a number of
mechanisms by which zinc interferes with the viral replication cycle. These include free virus inactivation (1), inhibition of viral uncoating
(2), viral genome transcription (3), and viral protein translation and polyprotein processing (4). No studies to date, however, have
demonstrated zinc-mediated inhibition of virus assembly and/or particle release. CV, coronavirus; DdDp, DNA-dependent DNA
polymerase; EMCV, encephalomyocarditis virus; FMDV, foot and mouth disease virus; HCV, hepatitis C virus; HIV, human immunodeﬁciency
virus; HPV, human papilloma virus; HRV, human rhinovirus; HSV, herpes simplex virus; PV, polio virus; RdRp, RNA-dependent RNA
polymerase; RT, reverse transcriptase; SARS, severe acute respiratory syndrome coronavirus; SFV, Semliki Forest virus; SV, sindbis virus; VZV,
varicella-zoster virus; Zn, zinc.
been comprehensively reviewed recently (135). As such, this
review will focus specically on the role of zinc in the
immune response to viruses.
Viral infections are recognized by a number of innate
immune receptors termed pattern recognition receptors
(PRRs). These include the cell surface and endosomal Toll-
like receptors (TLRs), as well as a variety of cytosolic
PRRs such as RIGI, MDA5, and IFI16 that primarily bind
viral nucleic acids (136). Following ligand binding, PRRs
share a number of downstream signaling intermediates,
that ultimately activate both inammatory (NF-κB, AP1)
and innate immune (IRF1/3/7) transcription factors. These
transcription factors cooperate to induce expression of IFNs,
ofwhichthereare3types:typeI(IFN-αand IFN-β), type
II (IFN-γ), and type III (IFN-λs). Type I and III IFNs
activate very similar antiviral signaling pathways; however,
the type I IFN response is ubiquitous, whereas the type III
IFN response is limited to a subset of immune cells, as well
as epithelial cells of the liver, gastrointestinal, and pulmonary
tracts (137). Although both IFN types bind unique receptors,
they activate a common signaling cascade where STAT1
translocation into the nucleus and subsequent binding of the
IFN-sensitive response element that is present in hundreds
of gene promoters. As stated previously, these ISGs possess
numerous roles including immune cell chemotaxis and
activation, as well as numerous antiviral mechanisms to
inhibit viral replication within infected and neighboring cells.
Zinc and pathogen recognition.
Upon recognition of microbial antigens by TLRs, a rapid
and transient inux of free zinc ions occurs. Interestingly,
this has been demonstrated in response to viral stimuli,
imiquimod, ssRNA40 (TLR7), and CpG (TLR9), but not
polyI: C (TLR3) in the mouse macrophage RAW 264.7 cell
line (138). In response to TLR7 activation, zinc was shown
to reduce the production of type I IFNs and ISGs CD80
and CD86. Based on results using other stimuli, the authors
suggest that zinc can inhibit IRF3-, and perhaps IRF7-
dependent IFNB production, by limiting activation and/or
nuclear translocation (138). The role of the zinc inux in
this context remains undened, but may reect a regulatory
mechanism to prevent excessive IFN production.
Although no direct inhibition of IRF signaling by zinc has
been demonstrated, zinc can modulate a number of factors
upstream of IRF activation. For example, the IκBkinase
(IKK) members IKKαand IKKβareinhibitedbyzinc,albeit
at high concentrations of ∼0.5 μM(139). IKKαhas been
shown to activate IRF7 in response to TLR7/9 stimulation
(140), whereas IKKβ(141), IKKεand TANK-binding kinase-
1 (TBK1) (142) can activate IRF3 following TLR3 stimuli.
Zinc can also stimulate expression of the deubiquitinating
enzyme A20 (43)toinhibitthepathogenresponse.A20
is a regulator of NF-κB- (143), TLR3- (144), and RIGI-
mediated (145) IFN production, most likely by targeting
PRR signaling components TIR-domain-containing adapter-
inducing interferon-β(TRIF), TNF Receptor Associated
Factor (TRAF) 2, and TRAF6. A20-decient cells are hyper-
responsive to viral infection, possess increased activation of
NF-κB, IRF3, and IRF7, and improved viral clearance (146).
Zinc and the interferon response.
After pathogen recognition, NF-κB, AP1, and IRF3/7 bind
IFN promoters to stimulate type I/III IFN production. Zinc
plays a signicant role in the response to IFNs by modulating
secretion, cytokine potency, and receptor binding, as well as
inuencing signaling intermediates and pathway inhibitors.
A recent study has demonstrated that intracellular zinc
can reduce IFN secretion by destabilizing sortilin mRNA
transcripts (147). Sortilin is an endosomal protein that
facilitates secretion of cytokines such as IFN-γand IL6 (148),
and its depletion results in a signicant reduction in secretion
of IFN-α. Consequently, because sortilin ensures tracking
and secretion of numerous cytokines, it is possible that zinc
also inhibits the secretion of other IFNs.
Structural studies have demonstrated that zinc ions can
mediate dimerization of IFNA molecules (149). Nonetheless,
generate despite using concentrated IFN (50 μM) and zinc
(1 mM). It is therefore likely that the circulating active
form of IFN-αis monomeric. A single study performed in
2001 showed that zinc can increase the antiviral activity of
IFN-α10-fold against rhinovirus challenge (150). Although
this study drew radical conclusions, antiviral activity was
been reproduced since. Moreover, zinc was added before
viral infection, which is known to interfere with rhinovirus
polyprotein processing (73,74), as reviewed above.
shown that zinc can inhibit IFN-λ3signaling,mostlikelyby
preventing receptor binding and subsequent signaling (28).
Upon demonstrating in 2014 that metallothionein expression
was IFNL genotype-dependent, and inversely associated
with ISG expression in HCV (151), we showed that serum
zinc was the driver of hepatic metallothionein expression.
Although zinc had minimal eect on IFN-αsignaling, it
could almost ablate IFN-λ3 signaling at a concentration of
50 μM, resulting in a signicant reduction in its antiviral
activity (28). Interestingly, we found no inhibition of IFN-
4, suggesting a highly specic
interaction. The mechanism by which zinc interferes with
the IFN:receptor interaction remains uncertain; however, we
have ruled out an eect of zinc on IFN-λ3disuldebond
Type I and III IFNs bind to unique receptor complexes
composed of IFN-αreceptors IFNAR1/IFNAR2 and IFN-λ
receptors IFNLR1/IL10RB, respectively, but signal via almost
identical pathways. Consequently, zinc may act to reinforce
the shared IFN signaling cascade by inhibiting protein
tyrosine phosphatase enzymatic activity (152). Following
receptor engagement by IFNs, intracellular Janus protein
tyrosine kinases Jak1 and Tyk2 become phosphorylated,
which in turn phosphorylate STAT molecules to stimulate
ISG expression. By dephosphorylating these key signaling
molecules, a number of phosphatases have been shown to
“put the brakes” on IFN signaling. Phosphatases tyrosine-
protein phosphatase non-receptor type 6 (SHP1), type 11
(SHP2), and protein phosphatase 2A (PP2A) have all been
shown to inhibit JAK-STAT phosphorylation (153–155),
and are all inhibited by zinc ions, predominantly in the
nanomolar range (156–158). Interestingly, PP2A can also
inhibit the phosphorylation of IRF3, thus regulating antigen
recognition by PRRs (159). Conversely, the tumor suppressor
phosphatase and tensin homologue (PTEN) stimulates IRF3
activation by removing inhibitory phosphorylation at Ser97
(160), and is also inhibited by zinc at nanomolar concen-
trations (161). Zinc inhibits numerous pro- and antiviral
phosphatases, with the net eect on virus recognition and
response being undened, which clearly requires further
To enable a highly controlled IFN response, negative
regulators of IFN signaling are often ISGs. These include
the suppressors of cytokine signaling (SOCS-1 and SOCS-
3), which bind and inhibit JAK protein signaling, thus
preventing signaling from numerous inammatory (IL-
6) and antiviral stimuli (162). Interestingly, zinc-driven
activation of the MTF-1 transcription factor can induce
expression of SOCS-3 in HepG2 cells (163). The zinc
importer ZIP-14, which is responsible for zinc inux
following inammatory stimuli, was required for SOCS-3
expression, and may represent yet another zinc-mediated
mechanism to limit the inammatory response. Although
the transporter responsible for hepatic zinc inux following
IFN stimulation remains unknown, it is perceivable that
ZIP-14 may drive zinc inux and subsequent SOCS-3
Zinc deﬁciency caused by disease, age, and lifestyle
factors: lessons from supplementation
Zinc status is primarily determined by dietary zinc intake;
however, additional factors such as dietary composition,
Zinc as an antiviral 9
alcohol intake, and disease state can signicantly reduce zinc
uptake and storage, or increase zinc excretion (164). With
respect to dietary composition, zinc supplementation as part
of a meal can signicantly reduce zinc absorption when
compared to water-based solutions of zinc (164). Moreover,
dietary phytate, a natural chelator of zinc ions that is present
in corn, rice, and cereals, can severely restrict zinc absorption
(165). Consequently, diets containing high phytate: zinc
molar ratios, can result in zinc deciency, even with adequate
zinc intake. Unfortunately, rural diets in low-income nations
are often zinc-poor and phytate-rich because of a dietary
reliance on rice and vegetables.
Aged individuals are also signicantly more susceptible to
zinc deciency, increasing their likelihood of acquiring life-
threatening viral infections (166). Ex vivo, zinc supplementa-
tion has been shown to improve leukocyte IFN-αproduction
(167) and to reduce mononuclear cell TNF production (168).
Year-long supplementation with 45 mg elemental zinc/d in
elderly subjects (aged 55–87 y), has also demonstrated a
dramatic reduction in the incidence of infection as well as
plasma oxidative stress markers (168).
Alcoholism can stimulate severe zinc deciency devel-
oped via numerous sociological and physiological mecha-
nisms, with factors including but not limited to 1)increased
urinary zinc excretion (169), 2) reduced zinc intake (poor
diet) (170), 3) reduced zinc absorption (171), and 4)anda
reductioninhepaticzincstores(172). Alcohol also stimulates
microbial dysbiosis and gastrointestinal permeability (173), a
phenotype that can increase the likelihood of viral infection
in the gut (174). Importantly, dietary zinc supplementation
can improve intestinal barrier dysfunction as a result of
alcohol and microbial infection (175,176).
As previously discussed, zinc deciency is common
among chronic infections such as HPV, HCV, and HIV (113,
123). Consequently, a number of studies have examined
the eects of zinc supplementation on antiviral immunity,
inammation, and treatment response. As described above,
zinc supplementation can improve HCV treatment response
and liver inammation caused by chronic infection. In
addition, long-term zinc treatment over 7 y has been shown
to reduce the risk of hepatocellular carcinoma progression in
chronic HCV patients, as assessed by multivariate analysis,
compared to controls (P<0.05) (105). Zinc supplementation
has also been assessed as an adjunct therapy to antiretroviral
administration in patients with HIV. One study reported
decreased diarrhea in patients treated with zinc compared
to controls (P<0.05 for both groups) (124). A more
recent study revealed an increase in CD4+Tcellcountin
patients treated with a combination of zinc and antiretroviral
therapy, compared to patients on antiretroviral therapy alone
(P<0.05) (177). Taken together, these data indicate that zinc
deciency is associated with greater disease activity in the
context of chronic viral infection. Oral zinc supplementation
may act in a synergistic manner when co-administered
with antiviral therapy and contribute to improved clinical
Vacc i n a t ion st u d ies .
Zinc supplementation during vaccination strategies has
provided an opportunity to examine the role of zinc in the
humoral response to viruses. A particular focus has been
applied to the eect of zinc supplementation on rotavirus
vaccination because of the high rate of mortality associated
with childhood diarrhea in developing countries. Unfortu-
nately, although zinc deciency is associated with increased
risk of rotavirus gastroenteritis (178), it does not greatly
increase the development of humoral immunity followed
by vaccination (rotarix), as dened by seroconversion rate
(179). Nonetheless, a pooled analysis of randomized trials
performed in 2000, demonstrated that zinc supplementation
shortens the length of diarrheal episodes and reduced the rate
of treatment failure or death by 42% in zinc-decient children
Comparable studies of supplementation with zinc before
vaccination have produced similar disappointing results.
Zinc supplementation did not improve seroconversion fol-
lowing administration of the oral poliovirus vaccine in
infants (181), nor did it improve the immunological response
to HBV (182) or inuenza vaccination (183) in the elderly.
Although there remains little evidence that zinc improves
viral vaccination responses, a small number of studies suggest
that zinc may improve antibody titers and antibacterial re-
sponses to pneumococcus (184)andcholerainfections(185).
Conclusions and Future Perspectives
The tight regulation of zinc homeostasis both systemically
and intracellularly indicates that zinc plays an essential
role in human health. Although zinc is a component of
compared with protein-bound) can stimulate a variety of
signaling events, including the antiviral response. In vitro
studies suggest that free zinc may possess potent antiviral
eects, and are supported by trials of creams, lozenges,
and supplements with high free zinc content. Moreover,
zinc-binding proteins such as the metallothioneins may
possess antiviral roles, although their specic function
remains uncertain. Nonetheless, zinc treatment applied at a
therapeutic dose and in the right form has the potential to
drastically improve the clearance of both chronic and acute
viral infections, as well as their accompanying pathologies
and symptoms. Consequently, the role of zinc as an antiviral
can be separated into 2 categories: 1) zinc supplementation
implemented to improve the antiviral response and systemic
immunity in patients with zinc deciency, and 2)zinc
treatment performed to specically inhibit viral replication
or infection-related symptoms (75,78–82,83,85–91,95–101,
The authors’ responsibilities were as follows—SR and GA de-
signed the review, all authors performed the study selection,
all authors read and approved the nal paper.
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