BGBiogeosciencesBGBiogeosciences1726-4189Copernicus PublicationsGöttingen, Germany10.5194/bg-18-4431-2021Ice nucleation by viruses and their potential for cloud glaciationIce nucleation by viruses and their potential for cloud glaciationAdamsMichael P.AtanasovaNina S.SofievaSvetlanahttps://orcid.org/0000-0001-5343-0870RavanttiJanneHeikkinenAinoBrasseurZoéhttps://orcid.org/0000-0001-5387-018XDuplissyJonathanhttps://orcid.org/0000-0001-8819-0264BamfordDennis H.dennis.bamford@helsinki.fiMurrayBenjamin J.b.j.murray@leeds.ac.ukhttps://orcid.org/0000-0002-8198-8131Institute for Climate and Atmospheric Science, School of Earth and
Environment, University of Leeds, Leeds, UKFinnish Meteorological Institute, Helsinki, FinlandMolecular and Integrative Biosciences Research Programme, Faculty
of Biological and Environmental Sciences, University of Helsinki, Helsinki, FinlandInstitute for Atmospheric and Earth System Research/Physics,
Faculty of Science, University of Helsinki, Helsinki, FinlandHelsinki Institute of Physics, University of Helsinki, Helsinki,
Finlandnow at: Institute for Molecular Medicine Finland, HiLIFE, University of
Helsinki, Helsinki, Finland
In order to effectively predict the formation of ice in
clouds we need to know which subsets of aerosol particles are effective at
nucleating ice, how they are distributed and where they are from. A large proportion of ice-nucleating particles (INPs) in many locations are likely
of biological origin, and some INPs are extremely small, being just tens of
nanometres in size. The identity and sources of such INPs are not well
characterized. Here, we show that several different types of virus particles
can nucleate ice, with up to about 1 in 20 million virus particles
able to nucleate ice at -20∘C. In terms of the impact on cloud
glaciation, the ice-nucleating ability (the fraction which are
ice nucleation active as a function of temperature) taken together with
typical virus particle concentrations in the atmosphere leads to the
conclusion that virus particles make a minor contribution to the atmospheric
ice-nucleating particle population in the terrestrial-influenced atmosphere.
However, they cannot be ruled out as being important in the remote marine
atmosphere. It is striking that virus particles have an ice-nucleating
activity, and further work should be done to explore other types of viruses
for both their ice-nucleating potential and to understand the mechanism by
which viruses nucleate ice.
Introduction
The formation of ice in clouds is critically important for the planet's
radiative balance and our prediction of future changes in climate with
increased greenhouse gas concentrations (Vergara-Temprado et al.,
2018; Tan et al., 2016). Ice-nucleating particles (INPs) have the potential
to cause supercooled liquid cloud droplets, present in mixed-phase clouds,
to freeze at temperatures greater than homogenous freezing, which can
drastically alter cloud properties such as albedo, composition and lifetime (Murray
et al., 2021; Hoose and Möhler 2012; Kanji et al., 2017; Hawker et al.,
2021). Despite the potential importance of INPs, there is still a lack of
knowledge regarding their characteristics, sources, and ultimately their temporal and spatial distribution around the globe.
Our current knowledge of atmospheric INPs (under mixed-phase cloud
conditions) suggests a number of potentially important aerosol types,
including mineral dust, marine organics and terrestrial bioaerosols (DeMott
et al., 2010; Kanji et al., 2017). The characteristics and source regions
for mineral dust are relatively better understood than other potentially
important INPs, and mineral dust from both high- (Sanchez-Marroquin
et al., 2020; Tobo et al., 2019) and low-latitude sources is thought to be
the dominant INP around much of the globe at temperatures <-20∘C. Marine organics and terrestrial bioaerosols have both been
demonstrated to play a major role in the global INP burden, but the nature
of these INPs is less well understood than that of mineral dust. Marine
organics are of particular importance in remote marine regions where there
is little mineral dust (Wilson
et al., 2015; Burrows et al., 2013). Terrestrial bioaerosols are thought to
outcompete mineral dust in the terrestrial mid-latitudes at temperatures
>-20∘C; however, their source(s) and nature are at
present poorly understood (Conen
et al., 2016; McCluskey et al., 2018a; O'Sullivan et al., 2018;
Vergara-Temprado et al., 2017).
Known INPs of biological origin include bacteria, fungi, pollen and marine organics amongst others (Kanji et al.,
2017). Bacteria and fungi exhibit ice nucleation due to the presence of
ice-nucleating proteins (Green
and Warren, 1985; Pouleur et al., 1992; Lindow et al., 1982), whilst the
ice-nucleating ability of pollen has been linked to polysaccharides
(Pummer et al., 2012;
Dreischmeier et al., 2017). Marine organic INPs, associated with sea spray,
are thought to be biogenic and are often smaller than 0.22 µm, but it
is currently not clear exactly what these ice-nucleating particles are, and
there may be multiple marine INP types (Creamean
et al., 2019; DeMott et al., 2016; Irish et al., 2017, 2019; Schnell et al.,
1975; Wang et al., 2015; Wilson et al., 2015). Compared to non-biological
INPs, some microorganisms such as specific bacteria or fungi nucleate ice at
relatively high temperatures; for example, the best-studied ice-nucleating
bacterium, Pseudomonas syringae (P. syringae), can nucleate ice at temperatures up to -2∘C (Morris et
al., 2004, 2013; Lindow et al., 1978). Despite the ice
nucleation potential of primary biological aerosol particles, recognized
since the 1970s (Schnell et al., 1976),
the global distribution and sources of biological INPs remain poorly
understood (Murray et
al., 2012; Kanji et al., 2017). Hence characterizing the ice-nucleating
ability of the various categories of biological aerosol particles is
important.
In bacteria, membrane proteins are thought to interact with water and impose
order in supercooled water in such a way as to promote nucleation of ice.
Pandey et al. (2016) demonstrated that
in the case of P. syringae patterned hydrophilic–hydrophobic regions due to the
interactions of amino acids belonging to the membrane protein led to the
increased ordering of water molecules coupled with efficient removal of
thermal energy from the surrounding water molecules into the bacterial cell.
This mechanism could potentially protect microorganisms at sub-zero
temperatures and preserve their viability and infectivity in the atmosphere
(Wilson et al., 2012; Morris
et al., 2013). Whether or not a bacterium has the potential to produce
ice-nucleating proteins is dependent on the presence of an ice nucleation
gene. At present, eight ice-nucleating proteins are known and reviewed in
the protein database UniProt, each with an associated gene (protein IDs:
O33479, P06620, Q47879, P16239, O30611, P09815, P20469, P18127). It is
thought that a single functional ice nucleation protein gene in bacteria is
both necessary and sufficient for ice nucleation activity. The ice nucleation activity (INA) of a
bacterium that has a gene for the ice-nucleating protein in its genome
depends on the expression of the gene (i.e. if the protein coded by the
gene is actually produced by the bacterium), the integration of the protein
into the outer membrane of the bacterial cell and stabilization of the protein complex by the surrounding membrane constituents.
Viruses are a presently under-studied source with respect to their potential
as atmospheric biological INPs. Very little is known about viruses in the
atmosphere in general, and even less is known about their potential to influence
cloud properties through cloud glaciation. The only studies we are aware of
in which the ice-nucleating ability of a virus was examined are those of
Junge and Swanson (2008), who studied the polar
Colwellia phage 9A, and Cascajo-Castresana et al. (2020), who
studied a series of common proteins and a single virus. The former found
that these virus particles did not nucleate ice in their experimental
system. The latter observed ice nucleation activity in the Tobacco mosaic virus (TMV), a plant virus that infects the family of Solanaceae such us tobacco,
tomato or pepper. TMV was shown to be above the baseline of the buffer
solution it was suspended in, and it was noted in the study that whilst TMV
had a lower onset freezing temperature than other samples in the study (a
range of proteins), when normalized to cumulative active site density it was
more active.
Compared to bacteria and other micron-sized, single-celled microorganisms,
viruses are considerably smaller (from ∼ 25 nm in diameter;
except for the nucleocytoplasmic large DNA viruses that are cellular size).
The small size of virus particles means that their atmospheric lifetime has
the potential to be on the order of many days to weeks in the atmosphere,
although this will depend on the size of the particles that they are
internally mixed with. This is considerably longer than the lifetime of
larger biological particles, especially those larger than ∼ 10 µm, which have lifetimes of only hours
(Grythe et al.,
2014; Reche et al., 2018) and therefore have atmospheric abundances which
decrease rapidly during transport (Hoose et
al., 2010).
In addition to supermicron entities such as bacteria, submicron-sized
biological particles have also been shown to be effective ice-nucleating
particles (O'Sullivan et al., 2015).
For example, it has been shown that there are biological INPs belonging to
fungal and pollen samples at sizes below 200 nm
(Pummer
et al., 2012; Fröhlich-Nowoisky et al., 2015). Fertile soil samples when
dispersed in water and filtered have also been shown to have a significant
number of ice-nucleating particles below 200 nm
(O'Sullivan
et al., 2015; Hill et al., 2016). O'Sullivan et al. (2015) showed that
some ice nucleation persisted in fertile soil samples filtered to 1000 kDa;
however, ice nucleation above -10∘C was removed by these
filtrations. Decayed plant litter was shown to have comparable INP
concentrations before and after filtration through 200 nm filter pores and
retained a fraction of these INPs when further filtered through 20 nm filter
pores (Vali et
al., 1976). Ice-nucleating particles below 200 nm were measured in North
American Arctic snow samples and in precipitation from North China temperate
grassland (Du et
al., 2017; Rangel-Alvarado et al., 2015). The snow samples were shown to be
of biological origin and subsequently tested for virus-like structures, of
which none was observed. Despite this, the authors stated they could not
preclude viruses as a potential explanation for the observed ice-nucleating
activity, based on the size of the INPs and their likely origin.
Measurements of INPs in the Arctic sea surface microlayer showed that most
of the observed ice nucleation (in the immersion mode) was caused by
particles between 0.02 and 0.2 µm in size and were heat labile;
viruses were suggested as a potential explanation
(Irish
et al., 2017; Wilson et al., 2015). Atmospheric measurements made in the
Arctic showed the presence of atmospheric INPs in the size range 150–340 nm (Creamean
et al., 2019; Creamean et al., 2018). Size-resolved measurements made in a
boreal forest in Hyytiälä, Finland, showed an instance in which INPs
in the size range 250–500 nm dominated the atmospheric INP burden at
temperatures >-22∘C, whilst measurements made at
near-surface-level locations in the UK showed INPs present at sizes below
250 nm (Porter et al.,
2020). There is a growing body of evidence that suggests there is a
reservoir of currently unidentified biological particles in the fine mode
(<250 nm) present in soil/plant life, the oceans and the
atmosphere. In this study, we test the hypothesis that viruses are a
potential candidate for the source of these fine-mode INPs.
It has been estimated that there are ∼ 1031 virus
particles in the biosphere (Whitman
et al., 1998), with approximately 107 virus particles per millilitre of
seawater, 108–109 per millilitre in marine surface sediments
(Suttle, 2007, 2005) and 108–109
per gram of soil in different types of terrestrial environments
(Srinivasiah et al., 2008). Numerous studies
indicate that there are approximately 10–100 times more viruses compared to
their host cells in any given environment (Srinivasiah et al., 2008;
Cai et al., 2019). With respect to viral abundance in the atmosphere, there
is at present a dearth of knowledge. Rastelli
et al. (2017) measured the viral abundance in both the seawater microlayer
and the aerosol phase directly above using a bubble generator system
designed to mimic wave breaking in open seawater. Virus concentrations for
seawater and sampled air were 5×1011 and 0.3–3.5 ×105 virus particles m-3,
respectively. Virus particles were measured from outdoor air samples using a
filter-based technique taken at a university campus, with the atmospheric
virus particle concentration being measured as 1.2±0.7×106 virus particles m-3 (Prussin et al., 2015). The spatial
and temporal variability of airborne viruses were investigated in a series
of different locations (residential district, forest and an industrial
complex), with concentrations of 1.7×106 to 4.0×107 virus particles m-3 being measured
(Whon et al., 2012). Overall, the
range of outdoor virus concentrations recorded in the literature range
between 0.3×105 and 4.0×107 virus particles m-3. It is likely that these numbers do not represent the full
variability of virus particle concentration due to the scarcity of
measurements.
Graphical representation of the virus particles used in the ice
nucleation study and their ice-nucleating ability. (a) Enveloped icosahedral
viruses. (b) Icosahedral viruses. (c) Pleomorphic viruses. (d) Lemon-shaped
virus. (e) Ice nucleation activity plots (expressed as nn), where hollow
markers indicate limit of detection (LoD) measurements in which the freezing
temperatures were consistent with the virus-free saline buffer control.
Virus particles are to scale according to the 100 nm scale bar. Temperature
values have been corrected for freezing point depression of NaCl.
Despite the large number of virus particles measured in various environments
there are a relatively small number of different particle structures a
virion (an infective virus particle) can have. This is due to physical
constraints of protein fold space that make up the virus particle
architecture (Abrescia et al., 2012).
Structurally similar viruses can have different host organisms and different
geographical source locations (Bamford
2003; Saren et al., 2005; Atanasova et al., 2012). There are several
observations of virus isolates with high genome identity originating from
spatially distant environments (Atanasova
et al., 2015; Pietilä et al., 2012; Saren et al., 2005; Tschitschko et al.,
2015). We have chosen virus particles for this study that represent several
different symmetric or asymmetric virus architecture types: icosahedral,
icosahedral with internal lipid membrane, icosahedral enveloped and
lemon-shaped (see Fig. 1a–e). As it would be beyond the realms of feasibility to test even a
minute fraction of the 1031 different viruses in the biosphere, we took
the approach that we believe allows us to investigate the maximum parameter
space and test the hypothesis that virus architecture/structure controls
ice-nucleating ability. In this study we present the ice-nucleating ability
of viruses with these different architecture types, demonstrate the
potential of different structural components in viruses to nucleate ice, and
attempt to estimate the potential of viruses as a class of atmospheric
ice-nucleating particles.
MethodsVirus growth, purification and production of Phi6 sub-viral particles
Virus particle suspensions were produced under carefully controlled
conditions which resulted in suspensions of high purity. The methodology for
producing virus particles has been developed over many decades and involves
first producing host bacterial cultures which are then infected with a
virus. In the case of lytic viruses, the virus causes the bacterial cells to
lyse (the cell membranes break down), which releases the contents of the
lysed cells (including virus particles) into the growth media. The growth
media solution then contains a mixture of components from the cells,
cell-wall fragments and virions. This cell debris is removed by
centrifugation, and the supernatant (containing the virus) is called the
lysate, which is the starting material used for virus purification. Notably,
in the case of non-lytic viruses, host cells release high numbers of virions
without lysis. In this case, the cells are removed similarly by
centrifugation, and the supernatant containing the viruses is used as the
virus stock solution. Nevertheless, for simplicity, in this study, the word
“virus lysate” is used to refer to the virus stock solution regardless of
whether the cells were initially lysed by the virus or not. In this study,
all other viruses are lytic, except for Salterprovirus His1 (His1), Alphapleolipovirus HRPV1 (HRPV1) and Alphapleolipovirus HRPV6 (HRPV6), which are
non-lytic. In order to examine the ice-nucleating ability of the virus
particles without the other components of the lysate, we employ some
state-of-the-art purification procedures for each virus, and these
purification protocols are described and referred to below. Shortly, the
virus particles in the lysate are precipitated using polyethylene glycol and
suspended into a small volume of buffer. The precipitate is cleared from
impurities by centrifugation and the viruses are purified (1×
purification) using rate-zonal centrifugation (in a sucrose gradient) where
the separation is based on the velocity of the virus particles. After the
purification, the light-scattering virus zone, which now has moved further
along the purifying gradient material, is extracted and further purified
(2× purification) using equilibrium centrifugation. This type of
purification is based on a density gradient, meaning that the virus
particles stop moving along the gradient when the density of the viruses
equals the density of the gradient medium. The 2× purified virus
sample is concentrated by differential ultracentrifugation, and the pellet
containing the 2× purified, concentrated virus sample is suspended
into a small volume of buffer. For some viruses, only 1× purification is
performed, after which the sample is concentrated similarly by differential
centrifugation. The concentrated, purified virus sample is used for the ice
nucleation activity assay (see below).
Bacterial and archaeal strains and viruses used in this study are listed in
Table S1 in the Supplement. Bacterial host strains were aerobically grown in Luria–Bertani
broth at 28 ∘C for P. syringae pathovar phaseolicola HB10Y and P. syringae LM2489, and at 37 ∘C for Escherichia (E.) coli HMS174 and E. coli C122 strains. Archaeal host strains were
aerobically grown in 23 % modified growth medium (MGM) at 37 ∘C (Nuttall and Smith, 1993).
Bacteriophages Salmonella virus PRD1 (PRD1) and Pseudomonas virus phi6 (Phi6) were 1× purified as described in
Bamford et al. (1995). The
2× purification of PRD1 was performed
(Bamford
et al., 1995; Lampi et al., 2018). The PRD1 particles devoid of DNA
(procapsids) were collected after 1× purification, during which the
DNA-containing particles sediment further along the sucrose gradient
compared to the empty procapsids. The 1× purified Phi6 was further
purified to 2× by equilibrium ultracentrifugation in 20 %–70 %
sucrose in 20 mM potassium phosphate buffer pH 7.2 with 1 mM MgCl2 (designated
here as potassium phosphate buffer) followed by concentration as described in
Bamford et al. (1995). Viruses Pseudomonas virus phi8 (Phi8),
Pseudomonas virus phi12 (Phi12), Pseudomonas virus phi13 (Phi13) and Pseudomonas virus phi2954 (Phi2954) were produced and precipitated according to
Qiao et al. (2010), and the 1×
purification was performed by rate-zonal ultracentrifugation in 5 %–20 %
sucrose gradients in potassium phosphate buffer, Sorvall AH629 rotor, 24 000 rpm, 50 min, 15 ∘C, followed by concentration using differential
ultracentrifugation, Sorvall T865 rotor, 34 000 rpm, 3 h, 10 ∘C.
All other viruses were purified to 1× preparations according to
protocols described in Eskelin et al. (2019)
(for PhiX174, Escherichia virus phiX174), Pietilä et
al. (2009) (for HRPV1), Pietilä
et al. (2012) (for HRPV6), Demina et al. (2016) (for HCIV-1, Haloarcula virus HCIV1)
and Bath et al. (2006) (for
His1).
Phi6 sub-viral particles were prepared according to
Bamford et al. (1995), modified by
Eskelin and Poranen (2018) (for butylated
hydroxytoluene treated particles). Phi6 nucleocapsids (NC) were prepared by
adding 1 % final concentration of Triton X-100 to 1× purified
Phi6 particles in potassium phosphate buffer and incubating 30 min at 22 ∘C. The treated particles were collected by ultracentrifuge, Ti1270 rotor,
30 000 rpm, 4 h, 15 ∘C. Particles were flushed three times with
and resuspended in 0.5 mL potassium phosphate buffer overnight at 5 ∘C.
The protein concentration of viral and sub-viral particles was measured by
the Bradford assay using bovine serum albumin as a standard
(Bradford, 1976). Virus samples were analysed by
sodium dodecyl sulfate 16 % polyacrylamide gel electrophoresis (SDS-PAGE)
(Olkkonen and Bamford, 1989) to visualize
viral protein profiles.
Search for ice nucleation motifs
Currently, there are eight referenced ice nucleation proteins identified
from bacterial cells according to the public protein database (UniProt,
https://www.uniprot.org/, last access: 7 September 2020). The ice nucleation motifs (INMs) predicted based
on these genes are short protein sequences conserved in this protein family.
They are abundant for the ice nucleation proteins (IN proteins) but scarce
in the rest of the bacterial genomes. The group of motifs specific for a
protein family can serve as a functional fingerprint indicating similarities
in structure and function. It was previously determined that INM3
corresponds to the clathrate structure part of the protein responsible for
ice nucleation activity in bacterial IN proteins
(Gurian-Sherman and Lindow, 1993;
Kajava and Lindow, 1993).
The INMs were acquired from SPRINT, an interface for the PRINTS data bank of
protein family fingerprints. SPRINT is a public domain database currently
maintained at the University of Manchester (http://130.88.97.239/dbbrowser/sprint/, last access: 28 June 2020). The INMs can be found in SPRINT
by the identifier ICENUCLEATN. All known ice nucleation motifs in IUPAC
(International Union of Pure and Applied Chemistry) nomenclature are listed
in Table S2. Since some of the putative viral proteins are not fully
characterized, we used protein INMs from SPRINT to build generalized
nucleotide motifs. The annotated viral genomes were acquired from the
National Centre for Biotechnology Information (NCBI) genome database (Table S3).
Ice nucleation motifs were searched for in the viral genomes using MEME
(Multiple Em for Motif Elicitation) Suite 5.1.0.
(Bailey et al., 2009). The search was
performed using MCAST (Motif Cluster Alignment Search Tool) and FIMO (Find
Individual Motif Occurrences) tools (Charles et al., 2011;
Bailey and Noble, 2003). MCAST searches for input motifs in the query
sequence for statistically significant clusters of non-overlapping
occurrences. FIMO, in turn, searches for individual motif occurrences in the
sequences and each motif independently. Each found occurrence was scored with
p value. The p score thresholds for significant findings were set to 0.0001.
In order to predict putative IN proteins in the viral genomes, total INM
coverage as well as the occurrence of repetitive IN motifs was studied. INM
coverage is calculated from the length of the matching INM sequence compared
to the total length of the protein. The INMs were annotated to the sequences
using Artemis 17.0.1, and the protein alignments were performed using Muscle
3.8.425 and visualized using Geneious Prime 2020.1.1. All the potential IN
proteins are listed in Table 1.
Potential IN proteins, their location, function and INM coverage.
Virus nameIN-protein candidatesProtein functionLocation in virusIN-motif coverage (%)Protein ID numberPhi6P3Adsorption to host cells, attachment to type IV pilusspikes18NP_620351.1P1Major inner capsid protein, RNA binding, replication and transcriptioncapsid20NP_620348.1Phi12P3cPutative host attachment proteinspikes16NP_690834.1P9Membrane proteinexternal lipid membrane28NP_690828.1Phi13P3bPutative host attachment proteinspikes31NP_690814.1P4Hexameric packaging NTPase5-fold vertices of the procapsid21NP_690818.1Phi2954P3Host attachment proteinspikes18YP_002600769.1PhiX174P11Minor spike proteinspikes29NP_040713.1HCIV-1Putative protein VP18Putative minor capsid proteinunknown hypothetically capsid24YP_009272867.1VP3Capsid proteincapsid31YP_009272848.1HRPV6ORF2Unknownunknown hypothetically capsid33YP_005454286.1ORF7Integral component of the membraneexternal lipid membrane47YP_005454291.1ORF8ATP-binding, AAA-type ATPaseunknown hypothetically capsid16YP_005454292.1PRD1P7Transclycosylaselipid membrane18YP_009639979.1P14DNA deliverylipid membrane31YP_009639980.1Ice nucleation experiments
Samples for analysis of the ice-nucleating activity of virus particles were
prepared by diluting 1× or 2× purified virus particles to
specific buffer solutions (Table S1) so that the final concentration of
plaque-forming units per microlitre (pfu mL-1) was 1010–1012. One plaque
corresponds to the progeny of one virus that initially infected the host
cell. Plaque-forming units measure the number of infective virus particles
in the sample. Sub-viral particles were used without dilution. Virus host
strains were collected by centrifugation (Eppendorf, 13 000 rpm, 5 min, 22 ∘C), diluted into the same buffer as the virus (Table S1),
centrifuged (Eppendorf, 13 000 rpm, 5 min, 22 ∘C) and resuspended
into buffer according to Table S1. NaCl (500 mM) was added to the buffer for
the archaeal cells and viruses due to them being classified as extreme
halophiles that require NaCl for optimum growth or infectivity.
Ice nucleation experiments were carried out using the µL-NIPI
(nucleation by immersed particles instrument) (Whale et al., 2015). In brief, the µL-NIPI analysis involved pipetting 1 µL droplets of sample suspension onto a hydrophobic-coated glass cover slip that was placed on top of an aluminium cold stage. Then, the cold stage was cooled until the droplets froze. Approximately 50 droplets were used per experiment, with temperatures ranging between 0 to
-36 ∘C. The cooling rate was 1 ∘C min-1. Viral
samples were agitated on a vortex mixer for 30 s prior to being
pipetted to ensure the particles were evenly distributed through the
suspension. Droplet freezing was recorded using a camera, with the freezing
temperature of each droplet recorded.
The cumulative fraction of droplets frozen on cooling to a temperature,
fice(T), is defined by
ficeT=niceTNtot,
where nice(T) is the cumulative number of droplets frozen on cooling to
T and Ntot is the total number of droplets. The cumulative number of
active sites per particle, nn(T), was calculated according to Eq. (2):
nnT=-ln1-ficeTnv,
where nv is the number of virus particles per 1 µL droplet. The
cumulative active sites per unit mass of material, nm(T), is defined by
nmT=-ln1-ficeTmv,
where mv is the mass of virus particles per droplet.
The freezing point depression of pure water due to NaCl (i.e. in the buffer
solutions) was calculated using
ΔTF=KF⋅b⋅i,
where ΔTF is the freezing depression, KF is the cryoscopic
constant (1.853 K kg mol-1 for water), b is molality and i is the Van 't Hoff
factor (2 for NaCl). Hence, we report the degree of supercooling relative to
the melting point of the aqueous saline solution. The correction was
typically about 1 ∘C for most virus suspensions (it was around
3 ∘C for an archaeal virus which required a very high salt
concentration).
ResultsIce-nucleating ability of virus particles
We studied virus ice nucleation from a virus structural perspective using
the nucleation by immersed particle instrument (µL-NIPI) technique
(Whale et al., 2015). We
examined the ice nucleation activity (INA) of 11 viruses with different
particle architectures, in an effort to probe the hypothesis that virus
architecture/structure influences the ice-nucleating ability of virus
particles (Fig. 1). These viruses included five enveloped cystoviruses of
P. syringae hosts with particle diameters of ∼ 85 nm (Phi6, 8, 12, 13 and
2954; Fig. 1a), two icosahedral viruses with an internal lipid membrane
and particle diameters of ∼ 70 nm (Fig. 1b, PRD1 and
HCIV-1), one of the icosahedral viruses without the DNA (Fig. 1b, PRD1 no
DNA), one 30 nm icosahedral virus without lipids (Fig. 1b, PhiX174), two
enveloped pleomorphic viruses with particle diameter of ∼ 50 nm (Fig. 1c), and one lemon-shaped virus (Fig. 1d). Phi6-like viruses
are commonly used as models for viruses that cause respiratory illnesses
like SARS-CoV-2, the causative agent of COVID-19, due to structure
similarity. Of the 11 viruses tested, 9 showed an INA distinct from
the INA of the buffer solution they were suspended in (Figs. 1e and S1 in the Supplement).
Phi12, an enveloped virus infecting P. syringae, was found to be the most ice-nucleation-active virus in our study (in terms of the number of INPs per
virus particle, nn). Phi12 was observed to trigger freezing from
-15 to -21 ∘C, with nn values between
5×10-10 and 5×10-8 per virus particle. The other structurally similar cystoviruses of P. syringae were all ice nucleation active, although less so compared to Phi12 (Fig. 1e).
At this point we address the question of if other components of the
bacterial cell lysate might account for the ice-nucleating activity reported
in this paper. We consider this possibility very unlikely for the following
reasons. (i) We have shown that the non-lysed host cells mostly have no
measurable INA (a few have a weak INA) (see Figs. S4 to S7). Several ice-nucleation-active P. syringae strains have been described in previous studies (de Araujo et al., 2019). None of the strains
contain functional genes that code ice-nucleating proteins – they only contain partial pseudogenes. (ii) Studies of ice
nucleation by bacterial cells which contain INA proteins and cells which are
lysed to some degree show that there are no measurable ice nucleation sites
that become active below about -12∘C (Wex et al.,
2015). Hence, the available evidence suggests that bacterial cell lysate
does not possess INA in the temperature ranges where we report activity in
this study. (iii) The purification steps described in the Methods section
remove the vast majority of the cell lysate material as demonstrated by the
protein profiles in Fig. S2. (iv) We show that a second purification
(2×), which would further remove any cell lysate material, has no
effect on the INA of the Phi6 sample. This is consistent with the INA being
related to the virus rather than the cell lysates. Hence, we conclude that
the activity we observe in our droplet freezing assays of purified virus
particle suspensions is most likely related to the virus particles.
The source of the INA was further studied using two of the
best-characterized model viruses, Phi6 of P. syringae and PRD1 of E. coli. Regarding Phi6, we
used biochemical dissociation to disassemble the virus particles into
sub-viral particles (Fig. 2). First, the virus spike proteins were removed
using butylated hydroxy toluene (BHT), with the resulting particle referred
to as Phi6 BHT and the separate spike proteins referred to as Phi6 P3.
Secondly, the lipid envelope and the associated proteins were removed using
the anionic detergent Triton X-100, exposing the nucleocapsid (NC) structure
of the Phi6 virion (Fig. 2a). Each of the sub-viral particles was shown to
have an INA distinguishable from the potassium phosphate buffer (Fig. 2b). Each of
the sub-viral components, along with Phi6, was normalized to the mass of
particles per volume of sample (nm). When normalized in this manner,
each component spanned approximately the same range in nm space,
102–104 (mg-1) across a range of temperatures. The
freezing spectrum of each component was similar, with the Phi6 BHT sub-viral
components having slightly warmer freezing temperatures than Phi6 at
equivalent nm values, whereas spike proteins (P3 in Fig. 2a) had a
slightly lower freezing temperature than Phi6. NC was found to be the
most IN-active sub-viral particle of Phi6 (Fig. 2c), freezing
approximately 4 ∘C warmer across the measured nm range when
compared to Phi6. INA is in part related to size (Pummer et al.,
2015). Since the spike proteins are only ∼ 20 nm in diameter,
whereas the BHT and NC particles are close to 80 nm, the difference in
activity may be related to size. It is not clear how virus particles behave
in the atmosphere, but several environmental stressors can disrupt virus
particles exposing their internal parts. The other Phi6 sub-viral particles
were also IN active (Fig. 2), indicating that the virus has broad IN
potential, either being active as a whole or in a disrupted form. PRD1 was
measured for its INA both with and without DNA. The nn values for PRD1
with and without DNA are shown in Fig. 3 and are similar to one another.
This result suggests that the presence of PRD1's DNA is not related to the
INA of the particles, indicating that also non-infective viruses (e.g.
viruses inactivated by different types of environmental stress) can have
INA.
Ice nucleation activity of the sub-viral particles of the Phi6 virus.
(a) Biochemical dissociation of the Phi6 virion. Small genome fragment is marked
as S, medium genome fragment as M and large genome fragment as L; P3 is the spike
protein, P5 is the lytic enzyme, P6 is the membrane fusion protein, P8 is the outer
capsid lattice protein and P9 is the major envelope protein; BHT means butylated
hydroxyl toluene; NC is the nucleocapsid. (b) Fraction frozen curves for Phi6 and
its sub-viral components. These values have not been correct for freezing
point depression due to NaCl. (c) The INA (expressed as active sites per unit
mass, nm) of Phi6 and its sub-viral components normalized to the mass of
particle per volume of suspension. Phi6 BHT in panels (b) and (c) refers to
spikeless enveloped icosahedral structure. Temperature values have been
corrected for freezing point depression of NaCl.
The number of active sites per particle (nn) for PRD1 with and
without DNA. Temperature values have been corrected for freezing point
depression of NaCl.
To further our understanding of the influence of virus structure on IN
activity, we tested six other viruses – four archaeal and two bacterial. Of
these six viruses, two archaeal viruses (HRPV1 and HRPV6) were enveloped
like Phi6 but lack particle symmetry and an NC structure (Fig. 1c). HRPV1
(Fig. S1b) was not distinguishable from the saline buffer (Table S1) it
was suspended in, whilst HRPV6 (Fig. S1b) was distinguishable from the
buffer but was not distinguishable from its host, and as such they are shown as
limiting values (Fig. S5). Viruses with icosahedral symmetry that contain
an internal lipid membrane (PRD1 and HCIV-1, Fig. 1b) were also tested to
further probe the dependency of viral INA on structure. PRD1, a well-known
model virus (Bamford et al., 1995), was
shown to be INA with a signal distinguishable from both the potassium phosphate
buffer and its host (Fig. S6) and nn values comparable to that of the
majority of the P. syringae viruses (excluding Phi12) (Fig. 1e). HCIV-1 did not have
an INA distinguishable from the saline buffer it was suspended in and is
thus shown as a limiting value (Fig. 1b). Another icosahedral virus,
PhiX174, this time without a lipid membrane (Fig. 1b), was tested and had
nn values similar to that of PRD1 and the majority of the P. syringae viruses. We
further studied the INA dependency on virus architecture by studying an
asymmetrical lemon-shaped archaeal virus, His1
(Bath et al., 2006).
Interestingly, the virus had a higher INA than all the tested viruses,
except for Phi12 (Fig. 1d–e), indicating that structurally different
viruses, symmetric or asymmetric, can be IN active. His1 was shown to be
distinguishable from the saline buffer solution (Fig. S1b) and its host
(Fig. S7).
Genetic analysis of ice-nucleation-active virus particles
The genomes of the 11 viruses included in this study were explored by
bioinformatic analysis to further examine the source of IN activity. The ice
nucleation activity observed in bacteria is due to protein structures which
mimic ice crystal clathrate structure on the cell surface, thus facilitating
ice crystal formation around the cell (Kajava and
Lindow, 1993). In viruses, the source of INA might also be of proteinaceous
origin. The possibility of the capsid or membrane proteins in virus
particles possessing similar structure and function to known bacterial ice
nucleation proteins as explanation for their ice nucleation capacity was
explored in this study. Proteins with potential ice nucleation activity were
screened by searching for conserved short sequences called ice nucleation
motifs in the amino acid sequence.
Viral proteins with significant INM coverage and presence of INM3 in their
sequence were predicted in eight of the viruses (Table 1). According to the
results, only Phi13 and PhiX174 did not have potential IN proteins, with
His1 having coverage below 15 % and so is not shown in the table. Other
viruses contained at least one potential protein with INM coverage of
15 %–50 % and obligatory INM3 presence. However, the INA of Phi13 and
PhiX174 is similar to the majority of the tested viruses such as Phi6.
Similarly, HRPV1 and HCIV-1 contain potential IN proteins, but these viruses
had the weakest INA of the tested virus particles. Therefore, the presence
of INMs in the sequence does not correlate to the capacity to nucleate ice.
Estimated viral INP concentration based on measured ice-nucleating
ability of virus particles and upper limit literature values of viral
particle concentrations in the atmosphere compared to measured INP
concentrations in both terrestrial (orange) and marine/polar (green)
environments. Table S4 shows a list of the studies from which the data to
create the field measurement envelopes were obtained. Temperature values
have been corrected for freezing point depression of NaCl.
Implications for the atmospheric ice-nucleating particle population
In order to estimate the INP concentrations associated with virus particles
in the atmosphere, we have combined the nn values shown in Fig. 1e and the
upper limit of the concentration of viruses in the atmosphere from
literature data (taken as 4×107 particles m-3; see
discussion in the introduction). It is important to note that we based these
virus INP concentrations on the INA of the specific samples which we studied,
and it may be possible that other virus particles have greater INA. However,
since there are a limited number of virus architectures and we test a range
of these architectures, we tentatively suggest that we capture the
typical range of INA of virus particles. Also shown on Fig. 4 are
envelopes showing the range of data from field campaigns in terrestrial
(orange) and remote marine/polar environments (green) (see Table S4 for a
list of representative measurements included in these envelopes).
As discussed in the introduction, in terrestrial environments, mineral dust
is thought to be a very important INP type, with marine organics playing a
secondary role (Vergara-Temprado et al.,
2017). In addition, there is evidence that biological INPs play an important
role in the terrestrial-influenced atmosphere in some locations (O'Sullivan
et al., 2018; Hill et al., 2016; Šantl-Temkiv et al., 2019; Conen et al., 2016; Pratt et al., 2009; Gong et al., 2020).
Figure 4 shows that across the entire temperature spectra relevant for
mixed-phase clouds, the concentrations of virus INPs are lower than the
lowest typical INP concentrations in terrestrial-influenced areas. The
closest the terrestrial envelope and the virus data points come to
overlapping is between -18 and -22 ∘C, at which
temperatures the difference in INP concentration is approximately 1 order
of magnitude, i.e. at most they might contribute about 10 % of the INP
population at around -20∘C. Furthermore, one might expect that
in environments where there is a strong source of virus particles there is
also a strong source of other biological materials; hence the influence of
virus INP may be overestimated in our simple analysis. Overall, these
results suggest that virus INPs generally play a minor role in regions
influenced by terrestrial INPs.
Remote marine locations are less influenced by active terrestrial sources,
and thus the INP populations there are different from those of the
terrestrial atmosphere (Creamean
et al., 2019; DeMott et al., 2016; McCluskey et al., 2018a, b). Marine organics and sea spray aerosol have been shown to be INP
sources of first-order importance in such environments (DeMott
et al., 2016; Vergara-Temprado et al., 2017; Wilson et al., 2015). Tests indicate that these INPs are sensitive to heat and can pass through 200 nm filters (Wilson et
al., 2015; Schnell and Vali, 1975), and while it is unclear exactly which
component of the seawater nucleate ice it has been pointed out that virus
particles are of the right size (Wilson et al., 2015). Field measurements
made in remote marine environments have reported remarkably low INP
concentrations. McCluskey et al. (2018a) measured
INP concentrations in a pristine marine environment at the Mace Head
research station in 2015, with INP concentrations as low as 10-3 L-1 at -20 ∘C. In a separate field campaign, measurements
were made in the Southern Ocean, and INP concentrations range between 3.8×10-4 and 4.6×10-3 at -20 ∘C (McCluskey
et al., 2018b). Figure 4 shows overlap between the virus INP data points and
the marine envelope in the temperature range from -15 to -27 ∘C, with the most active of the virus INPs being approximately
15× higher than the lower limit of the marine envelope at -20 ∘C. Whilst this by no means proves that virus INPs are important
in remote marine environment, it indicates they may contribute to the
atmospheric INP burden in such regions. However, the lowest INP
concentrations in the remote marine environment are most likely associated
with periods when the aerosol concentrations were lowest, as a result of the
combined effect of precipitation scavenging and weak sources. Under these
conditions, virus particles would also presumably be depleted.
Summary and conclusions
In this study we show that a range of viruses can nucleate ice
heterogeneously when immersed in supercooled solution droplets. A selection
of virus types with diverse architectures are shown to have ice-nucleating
abilities spanning 3 orders of magnitude at -20 ∘C, when
normalized to particle number. We probed the virus ice-nucleating ability
dependence on virus particle structure/architecture, showing that for our
selected viruses there was not a dependency on certain virus architecture.
Bioinformatic analysis shows that our current knowledge of ice nucleation
related to ice
nucleation protein genes in bacterial ice nucleators is likely
insufficient to understand why viruses nucleate ice, which can be due to
e.g. the overall arrangement of structural proteins making up the virion.
Our results are based on a small subsample of virus types but include
several of the most prominent viral architectures. Nine out of 11 tested
viruses were ice nucleation active, indicating that several structurally
different viruses can have IN potential. In addition, it has been shown
previously that a helical virus, Tobacco mosaic virus (TMV), can also
nucleate ice (Cascajo-Castresana et al.,
2020). While we have selected virus particles with a range of architectures
that are relatively common in nature, it is possible that other virus
particles nucleate ice more or less effectively. In particular, the specific
virus types we have studied here are from the terrestrial and freshwater
aquatic environment; the isolation and testing of a range of marine viruses
presents an important next step in quantifying the importance of viral ice
nucleators. More work needs to be done to understand what drives viral ice
nucleation and whether it would be dependent on virus structure/morphology,
host or some other factor.
This study shows the potential role viruses play as atmospheric INPs in
certain environments. The ubiquity of viruses in the atmosphere implies they
could serve as a baseline of INPs in situations where other, better-known
atmospheric INPs are absent in any meaningful quantity. However, our
estimates for the upper limit of virus INPs suggest they do not play a
meaningful role in terrestrial environment but may contribute to the INP
population in marine environments. More work needs to be done to understand
both why viruses nucleate ice and what role they play in regional and
global atmospheric ice nucleation.
Data availability
The data associated with this paper are openly available from the University
of Leeds data repository (10.5518/1019, Adams et al., 2021).
The supplement related to this article is available online at: https://doi.org/10.5194/bg-18-4431-2021-supplement.
Author contributions
MPA, NSA, SS, AH and ZB performed the experiments. SS designed
and performed bioinformatic analyses. DHB developed the NC purification
protocol. MPA, NSA, BM and DHB designed the experiments. All authors
participated in data analysis and interpretation of results. NSA, JD,
JR, BJM and DHB supervised and supported the project. The manuscript
was written by MPA, NSA, SS, BJM and DHB. All authors reviewed and
approved the manuscript.
Competing interests
The authors declare that they have no conflict of interest.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
Helin Veskiväli and Emeline Vidal are thanked for technical assistance.
We thank Leonard Mindich for providing bacteriophages Phi12, Phi13,
Phi2954 and the P.syringae bacterial strain. Ben Fane is thanked for providing
the PhiX174 bacteriophage. The use of the facilities and expertise of the
Instruct-HiLIFE Biocomplex unit, member of Biocenter Finland and
Instruct-FI, is gratefully acknowledged.
Financial support
This research has been supported by the European Research Council (CryoProtect (grant no. 713664) and MarineIce (grant no. 648661)), the Natural Environment Research Council (grant no. NE/T00648X/1), and the Academy of Finland (grant no. 309570). Nina S. Atanasova was supported by
the Academy of Finland postdoctoral grant 309570 and the
Scientific Advisory Board for Defence grant VN/627/2020-PLM-9.
Review statement
This paper was edited by Paul Stoy and reviewed by two anonymous referees.
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