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J. Biosci., Vol. 6, Number 4, October 1984, pp. 569–583
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How viruses damage cells: alterations in plasma membrane function
C. A. PASTERNAK
Department of Biochemistry, St. George’s Hospital Medical School, Cranmer Terrace,
London SW 17 ORE, UK
MS received 13 August 1984
Abstract. The effect of viruses on plasma membrane function has been studied in two types of
situation: (i) during the toxin-like action of paramyxoviruses when fusing with susceptible
cells, and (ii) during an infectious cycle initiated by different viruses in various cell types.
The nature of the permeability changes induced during the toxin-like action of viruses, and
its modulation by extra-cellular Ca2+, are described: membrane potential collapses, intracellular ions and metabolites leak out of, and extracellular ions leak into cells, but lysis does not
take place. The biological significance of such changes, and their relation to changes induced by
other pore-forming agents, are discussed.
Changes in membrane permeability such as those mentioned above have not been detected
during infection of cultured cells by paramyxo (Sendai, measles, mumps), orthomyxo
(influenza), rhabdo (vesicular stomatitis), toga (Semliki Forest) or herpes viruses. On the
contrary, sugar uptake is increased when BHK cells are infected with vesicular stomatitis virus,
semliki forest virus or herpes virus. Cultured neurones infected with herpes simplex virus show
changes in electrical activity. The pathophysiological significance of these alterations in
membrane function, which occur in viable cells, is discussed.
It is concluded that clinical symptoms may result from cell damage caused by virally induced
alterations of plasma membrane function in otherwise intact cells.
Keywords. Ca
2
+
; permeability; plasma membrane; viruses.
Introduction
The plasma membrane is the first part of a cell with which a virus comes in contact when
invading tissues and much of the host cell specificity of viral action is due to binding
between particular components at the cell surface and proteins on the surface of the
virus. Entry of the viral genome which follows, is generally by endocytosis of the intact
virus; the genome is released into the cytoplasm following breakage of the endocytic
membrane. In the case of paramyxoviruses, a family of enveloped RNA viruses that
contain a glycoprotein in their surface capable of initiating membrane fusion at neutral
pH, the viral genome is introduced directly into the cell as a result of fusion between the
viral envelope and the cell plasma membrane (figure 1).
The plasma membrane is usually involved a second time following uncoating and
expression of the viral genome inside the cell. In the case of enveloped viruses that are
released by budding from the plasma membrane, proteins of the viral envelope become
inserted into the membrane prior to budding. For other viruses also, virus-coded
Abbreviations used: BHK, Baby hamster kidney; dGlc, deoxyglucose; VSV, vesicular stomatitis virus; SFV,
Semliki Forest virus; HSV, herpes simplex virus; MeGlc, methyl glucose.
B
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Pasternak
Figure 1. Events during the infection of cells by viruses.
proteins appear at the cell surface and this forms the basis of the immune response to
viral infection. Moreover, new host-coded proteins may appear at the cell surface, and
existing proteins become modified, with or without triggering an immune response; the
transforming viruses are an example of this situation.
During acute infections by cytolytic viruses, especially by the non-enveloped ones, in
which the infected cell finally bursts as large amounts of virus are released, the plasma
membrane becomes damaged to the point of rupture. At this time, cells become
permeable to trypan blue, to cytoplasmic proteins which leak out, and to ions such as
Na+ and Κ+ (the capacity of the Na+ pump to reverse this trend having been exceeded).
The latter event leads to the entry of water and to cell swelling, and this is a contributing
factor to the lytic action of these viruses.
In terms of the pathophysiological consequences of viral action it is generally
assumed that cell lysis, leading to tissue necrosis, is the underlying cause (figure 2). Yet
there is no more reason for assuming that cell death is necessarily responsible for the
symptoms of viral infection, than there is in the case of diseases such as juvenile diabetes
or the haemoglobinopathies: in these cases the symptoms of the disease are due to an
alteration in the function of muscle or blood cells respectively, not to their destruction.
Such considerations have led the author to pose the question that forms the basis of this
article: to what extent do virally-mediated alterations of plasma membrane function
Figure 2.
Pathogenesis of viral disease.
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571
underlie changes in cell behaviour? We have examined two situations: the first is that of
viral entry; the second is that of alterations during the early stages of the infectious
process (which may bear similarity to the case of a persistent infection). The third
situation, that of events just prior to cell lysis, is pertinent to the induction of cell death,
rather than to that of cell damage (figure 2), and will not be discussed further. Several
authors have not distinguished sufficiently critically between the second and third
situation: a partial leakage of ions, for example, has been interpreted as being distinct
from cell lysis (Carrasco and Lacal, 1984); yet this may reflect no more than the fact that
lysis of cells is not synchronous, so that an apparent partial leakage of ions is actually
due to complete leakage (lysis) from some cells, and no leakage from others.
Viruses as toxins
Toxins may be defined as substances that damage cells in vitro, and as a result cause
disease in vivo. Certain constituents of the venom of wasps, spiders or snakes are
examples, as are the proteins produced by gram negative and gram positive bacteria
(figure 3). In the case of viruses, the postulate is that the viruses themselves are the
toxins. Just as cholera, diptheria or botulism results from an interaction between
bacterial proteins and cells without the occurrence of an infectious cycle (figure 3), so it
is suggested that clinical symptoms result from interaction between certain viruses and
susceptible cells without the participation of an infectious cycle (figure 4). Haemolytic
paramyxoviruses are obvious candidates for study. This is because during the entry
process, achieved by fusion between viral envelope and cell plasma membrane, the cell
becomes leaky to the extent that, in erythrocytes, lysis (i.e. haemolysis) ensues. Since
lysed cells are no longer viable, the situation is one of cell death, not cell damage. The
reason for discussing haemolytic paramyxoviruses in the present context is that in cells
other than erythrocytes, lysis is often not the end result, and instances will be described
in which changes in cell permeability lead to a transient alteration of cell behaviour.
Figure 3.
Action of bacterial toxins.
Figure 4.
Toxin-like action of viruses.
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Permeability changes induced by haemolytic paramyxoviruses
The system studied most extensivley to date is that of Sendai virus interacting with
Lettre cells (a line of malignant mouse ascites cells akin to Ehrlich ascites cells). Of other
viruses tested so far, only Newcastle Disease virus acts in a similar way (Poste and
Pasternak, 1978; Foster et al., 1980), though other enveloped viruses (such as influenza)
do so if the pH is reduced to below 6 (Patel and Pasternak, 1983). The specificity with
regard to cell type is much less, and every cell so far tested (except sheep erythrocytes;
unpublished observation) responds to Sendai virus (Pasternak, 1984); this is because
the requirement for viral binding is no more specific than the presence of a sialic acidcontaining glycoprotein (Scheid, 1981), and most cells possess such molecules at their
surface.
The events that occur when virus is added to cells are summarized in figure 5. Binding
between virus and cells is a temperature-independent process. It is followed by fusion
between the viral envelope and the cell plasma membrane; membrane fusion is a highly
temperature-dependent process, with a Ql0 of approximately 7 between 7 and 37°C
(Micklem et al., 1984a). Membrane fusion commences without a lag (Micklem et al.,
1984a), though the onset of permeability changes is characterized by a temperaturedependent lag period (Pasternak and Micklem, 1973; Micklem and Pasternak, 1977);
lag appears to reflect the build-up of a sufficient amount of membrane damage to be
manifest as changes in properties such as surface membrane potential (Okada et al.,
1975; Fuchs et al., 1978; Impraim et al., 1980; Bashford et al., 1983a,b), permeability of
monovalent (Fuchs and Giberman, 1973; Pasternak and Micklem, 1974a; Poste and
Pasternak, 1978; Bashford et al., 1983a, b) and divalent (Getz et al., 1979; Impraim et al.,
1979; Fuchs et al., 1980; Hallett et al., 1982) cations, permeability of phosphorylated low
molecular weight metabolites such as phosphoryl choline (Pasternak and Micklem,
1973), sugar phosphates (Pasternak and Micklem, 1973), nucleotides (Impraim et al.,
1980), low molecular weight peptides (Wyke et al., 1980), and so forth. The cut-off point
appears to be at a molecular weight of approximately 1000, so that proteins and other
macromolecules do not leak across cells (Pasternak and Micklem, 1973; Poste and
Pasternak, 1978), except at high doses of virus (Tanaka et al., 1975; Yamaizumi et al.,
1979). That is why these viruses, though haemolytic, are not cytolytic (Knutton et al.,
1976). Lag, that is the acquisition of a threshold number, or size, of permeability pores is
shorter for membrane depolarization than for leakage of ions, which is shorter than for
leakage of phosphorylated metabolites; thus the onset of permeability changes is
sequential (figure 6). Since cells recover from the effects of virus, through membrane
turnover, through lateral dispersal of protein ‘pores’, and through other mechanisms, it
Figure 5. Events induced by haemolytic paramyxoviruses.
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573
Figure 6. Sequential onset of permeability changes.
is possible under certain conditions to observe membrane depolarization without ever
initiating leakage of phosphorylated metabolites, for example (Pasternak, 1984).
Modulation of permeability changes: Ca2+ and Ca2+ antagonists.
Some 30 years ago it was noted that haemolysis by Newcastle Disease virus (Burnet,
1949), mumps virus (Morgan, 1951) and Sendai virus (Fukai and Suzuki, 1955) is
prevented by high concentrations of extracellular Ca2 +. Since then, the inhibitory effect
of Ca2+ at physiological concentrations (i.e. mM) on Sendai virus-mediated permeability changes has been demonstrated in a number of systems (Pasternak and
Micklem, 1974a; Pasternak et al., 1976; Impraim et al., 1979; Foster et al., 1980; Forda et
al., 1982). Ca2+ exerts its action in several ways, all of which may be the result of an
interaction with one particular type of receptor at the cell surface: the lag period to
onset of permeability changes is lengthened (i.e threshold is increased), the leakage of
ions and metabolites across the membrane is partially inhibited, and recovery of cells is
accelerated (Impraim et al., 1980). Although the amount of Ca2+ required to prevent
permeability changes is proportional to the amount of virus added, Ca2+ acts neither to
prevent virus-cell binding (Wyke et al., 1980), nor to prevent virus-cell fusion
(Pasternak, 1981). How it does act remains to be established: while there is a similarity
with communicating junctions in that, these too, are blocked by Ca2+, the concentration of Ca2+ required in that case is in the micromolar (Unwin and Ennis, 1984), not
millimolar range, since it acts intracellularly (Rose and Loewenstein, 1975); moreover
Mn2+ is as effective as Ca2+ in blocking virally induced permeability changes, [though
Mg2+ is not (Impraim et al., 1979)] whereas Mn2+ is ineffective at closing
communicating junctions.
In order to try and understand the mechanism by which Ca2+ affects permeability
changes, we have examined the effect of a number of drugs known to block the action of
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Pasternak
Ca2+ in excitable cells (Fleckenstein, 1977; Henry, 1980). Surprisingly, these drugs
proved to have an anti-Ca2+-like action in Lettre cells also. Thus verapamil, D600 and
prenylamine shorten lag, and increase the extent of leakage; in the presence of a
concentration of Ca2+ which on its own would inhibit leakage entirely, the addition of
prenylamine allows leakage of ions and phosphorylated metabolites to occur. The
drugs do not affect leakage itself, in contrast to Ca2+ chelators such as EGTA. Other
types of compound which we tested are inhibitors of calmodulin, such as trifluoperazine and calmidazolium (R24571): these proved to have an action similar to the Ca2+
antagonists; they are active at the same concentrations (10–5 Μ trifluoperazine and
10–6 Μ calmidazolium) as those at which they inhibit calmodulin-activated enzymes
such as phosphodiesterase (Van Belle, 1981). It has been postulated that Ca2+
antagonists such as verapamil, D600 and prenylamine bind to calmodulin at the same
hydrophobic site as trifluoperazine and calmidazolium (Johnson and Wittenauer,
1983). When the binding constants of all these drugs were compared with the
concentrations of drug required to achieve 50 % stimulation of leakage, the two sets of
data were found to be in reasonable agreement (Micklem et al., 1984b), suggestive of a
role of calmodulin in protecting the cell surface against virally-mediated permeability
changes (figure 7).
Figure 7. Role of Ca2+ in protecting against virally-mediated permeability changes.
The broken line indicates a virally-induced pore; Ca2+ (a) prevents the induction of pores,
(b) inhibits leakage through pores and (c) accelerates dispersal of pores (‘recovery’). Direct
evidence for the participation of calmodulin has not yet been obtained; the reason for
2+
implicating it is merely that inhibitors of calmodulin have an anti-Ca like action.
From Micklem et al. (1984b).
A word of caution is, however, necessary. First, a detergent like triton XI00 has an
action, at a concentration well below its critical micellar concentration, similar to the
Ca2+-antagonist drugs; it should be emphasized that all the agents mentioned affect
permeability changes at a concentration at which they are without effect in the absence
of virus. While there is no reason why an aromatic compound such as triton should not
bind to the same site as the (aromatic) Ca2+-antagonists, the same might not be
expected of a non-aromatic detergent like lubrol; yet lubrol is as effective as triton XI00
(unpublished experiment suggested by Dr Carlos Gitler). Second, the Ca2+ antagonists
are much more effective at stimulating permeability changes at low temperature (e.g.
20°C), than at 37°C. This suggests that the drugs might affect membrane fusion (see
above). On the other hand fusion is not a Ca2+ -sensitive event; moreover the induction
of permeability changes by purified preparations of the bee venom protein mellitin (see
below) is also susceptible to stimulation by calmidazolium at low temperature. Since
membrane fusion does not occur in this instance the drugs are more likely to have a
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General ‘membrane-weakening’ effect. Indeed, there is accumulating evidence that the
action of calmodulin inhibitors is not as specific as previously thought (Landry et al.,
1981; Corps et al., 1982; Gomperts, 1984). Hence it is premature to draw any
conclusions other than that Ca2+ protects cells against virally-induced permeability
changes, and that detergents and drugs that happen to have an anti-Ca2+-like action
potentiate permeability changes.
Similarity to other pore forming agents
If the action of haemolytic paramyxoviruses on cells is due to the induction of some kind
of hydrophilic pore in the plasma membrane, then the sequence of events described
above,—namely membrane depolarization, leakage of ions, leakage of phosphorylated
metabolites,—is likely to occur during the generation of pores by other toxin-like
substances also. We have examined three types of pore-forming agents, and each
exhibits just such a response at sub-lytic concentration: the protein mellitin of bee
venom (Haberrnan, 1972), the α-toxin of Staphylococcus aureus (Arbuthnott, 1982) and
the factors of activated complement (Mayer, 1972; reviewed by Bhakdi and TranumJensen, 1983, 1984 and by Muller-Eberhard, 1984). In each case extracellular Ca2+
inhibits the response; in no case, of course, is membrane fusion involved. Higher
concentrations of Ca2+ are required to prevent permeability changes, especially those
induced by S. aureus α-toxin, than by Sendai virus, and in the case of α-toxin, Mg2+ is as
effective as Ca2+ (Bashford et al., 1984a). Sensitivity to inhibition of pore-formation by
divalent cations clearly depends both on the cell type under study (Lettre cells are more
sensitive than erythrocytes, for example) and on the nature of the agent used. The latter
observation is hardly surprisingly in view of the fact that the effective pore size varies,
from approximately 1 nm diameter (Sendai virus; Wyke et al., 1980), to 2–3 nm (αtoxin; Fussle et al., 1981) to 0·9–7 nm (complement, depending on the amount of C9;
Bhakdi and Tranum-Jensen, 1984). Despite such differences the overall similarity in
response to extracellular Ca 2 + makes study of these pore-forming agents in relation to
the toxin-like action of viruses worthwhile.
Biological significance of permeability changes
A change in the permeability properties of excitable cells is likely to affect their
function. It is not surprising, therefore, that Sendai virus causes cultured neurones to
lose excitability, cultured heart cells to stop beating, and isolated pituitary cells to
secrete ACTH and other hormones (Forda et al., 1982; Pasternak and Micklem, 1984).
The effect is presumably caused by membrane depolarization and/or Ca2+ entry. Such
experiments have to be conducted at sub-physiological concentrations of Ca2+; at
higher concentrations of Ca2+ the effects are abolished (see above). The interesting
point is (a) the rapidity of the response (a change in membrane potential of neurones
within seconds of adding virus) and (b) the transient nature of the response (complete
restoration of neuronal excitability, of heart beat, or of cessation of hormone release,
within minutes). In short, the physiological function of the respective cells is impaired
without loss of viability (see figure 2), and with recovery of the original function. Such
situations provide clear illustrations of how the toxin-like action of viruses can lead to a
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Pasternak
transient alteration of cell behaviour. To what extent they contribute to clinical
symptoms during viral infections is an important topic for future investigations.
One area where virally-induced permeability changes might play a role is in the effect
of viruses on neutrophils and macrophages. An impairment of neutrophil function, for
example, has been suggested to be responsible for the secondary bacterial infections
that often follow a primary infection of the respiratory tract by viruses such as influenza
(Larson and Blades, 1976). Using luminol-induced chemiluminescence as an indicator
of neutrophil function (namely the generation of oxygen radicals within cells), however,
we have found that permeability changes underly neither the action of influenza virus,
nor that of Sendai virus, on neutrophils (S. Mehta and C. A. Pasternak, unpublished
results).
Virally induced permeability changes also have a role in biological research. First, the
use of Sendai virus to mediate cell-cell fusion and the generation of hybrid cells (Harris,
1970) depends on cell swelling brought about by the increased permeability to ions
(Knutton and Pasternak, 1979). Second, the generation of a permeability pore of
approximately 1 nm allows the introduction into cells of molecules of < 1000 daltons
such as cyclic nucleotides, EGTA and similar compounds that bind Ca2+, etc. The latter
type of agent has been used to introduce Ca2+ at micromolar concentrations into mast
cells (Gomperts et al., 1981, 1983) and pituitary cells (Gillies et al., 1981; Pasternak,
1984), in order to assess what concentration of Ca2+ is required intracellularly in order
to trigger histamine release and ACTH release, respectively, from these cells. The fact
that virally-induced permeability changes are transient, and can be controlled by
extracellular Ca2+, makes this method of permeabilizing cells preferable to techniques
employing detergents (Miller et al., 1979) or electric shock (Baker and Knight, 1978).
Surface changes during viral infection
We have measured three surface properties during the course of an infectious cycle
initiated by a number of different viruses: permeability of various cells to ions and low
molecular weight compounds, sugar transport by baby hamster kidney (BHK) cells and
excitability of cultured neurones.
The reason for the first investigation is obvious. Having demonstrated that certain
paramyxoviruses induce a permeability change when entering cells by membrane
fusion, it was of interest to examine whether similar changes occur when these and
other enveloped viruses are released from cells at the end of an infectious cycle by what
is essentially the reverse process, namely ‘budding’ from the cell surface. Moreover it
has been suggested not only that permeability changes do occur during the maturation
of various viruses (Carrasco, 1977, 1978; Carrasco and Lacal, 1984), but that such
changes underly the mechanism by which cells switch from the synthesis of host
proteins to that of viral proteins (Carrasco and Smith, 1976, 1980) (figure 8).
The second investigation arose from the first. We were using [3H]-deoxyglucose
(dGlc) uptake by BHK cells as a measure of an increased permeability to low molecular
weight compounds such as sugar phosphates, and found an unexpected result: instead
of taking up less [3H] (as cells permeabilized with Sendai virus, for example, do, since
[3H]-dGlc-6-P formed intracellularly immediately leaks out again; Impraim et al.,
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577
Figure 8. Hypothesis of Carrasco and Smith (1976, 1980).
1980), BHK cells infected with a number of different viruses were found to take up more
[3H]. The basis for this apparently anomalous effect has now been explored in some
detail.
The third investigation was triggered by the fact that herpes viruses are known to
infect ganglionic neurones and, in the case of a herpes virus such as Varicella zoster
(chicken pox virus), may remain latent there for many years; a study of the electrical
properties of the cell surface of herpes simplex virus (HSV)-infected neurones yielded
another unexpected result, and this system was therefore investigated further.
Membrane permeability
In contrast to the permeability changes induced by the toxin-like action of paramyxoviruses, changes induced during viral budding from cells, or at some earlier stage
of the infectious cycle, are likely to be more stable, and to appear at some characteristic
time after the establishment of the viral genome within the cell (figure 9). Infection of
various cell types, grown in monolayer or in suspension culture, with myxoviruses such
as Sendai, measles, mumps or influenza virus (Foster et al., 1983), with a rhabdovirus
(vesicular stomatitis virus, VSV), a togavirus (Semliki Forest virus, SFV) (Gray et al.,
1983a) or a herpes virus (HSV) (Μ. Η. James and C. A. Pasternak, unpublished results),
have not revealed any permeability changes similar to those described in the first part of
this article. True, there does appear to be a decreased concentrative uptake of sugars
and amino acids (comparable with an increase in membrane permeability; Pasternak
Figure 9. Permeability changes induced by viruses.
A. Toxin-like changes. B. Changes during an infectious cycle.
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Pasternak
and Micklem, 1974b; Impraim et al., 1980) by certain cells infected in suspension culture
(Pasternak and Micklern, 1981), but the effect is much smaller than that induced by the
toxin-like action of haemolytic paramyxoviruses; moreover the decreased uptake of
nutrients is not accompanied by leakage of monovalent cations down their concentration gradients (Pasternak et al., 1982; Gray et al., 1983a; Foster et al., 1983), as would be
expected if the permeability barrier of the cell had been breached by the creation of a
hydrophilic pore. And although the membrane potential of some cells appears to
decrease after infection with certain viruses (Pasternak and Micklem, 1981; Pasternak
et al., 1982; Bashford et al., 1984b), this is not the result of a generalized increase in
membrane permeability.
In one instance, namely in SFV-infected BHK cells, a modest increase in intracellular
Na+ does occur, though without any decrease in intracellular K+ (Gray et al., 1983a).
Since it is an increase in intracellular Na+ that has been postulated to account for the
shut-down of host protein synthesis (Carrasco and Smith, 1976,1980; Garry and Waite,
1979; Garry et al., 1979, 1982), this situation was examined further. It was found that a
greater increase in intracellular Na+, brought about either by the addition of the
Na+/K+ ionophore nigericin or by a brief exposure of cells to haemolytic Sendai virus,
resulted in a much lesser effect on protein synthesis than did infection with SFV (Gray
et al., 1983b). Hence even in this case an altered intracellular Na+ concentration
(whether achieved as a result of a permeability change or not) cannot adequately
account for the shut-down of host protein synthesis.
Another aspect of Carrasco’s hypothesis (figure 8), that infected cells become
permeable to low molecular weight compounds (Carrasco, 1977), is the following.
Infected cells might be expected to take up low molecular weight inhibitors of protein
synthesis, such as the GTP analogue GppCH2p, better than uninfected cells, and as a
result of impaired protein synthesis become so incapacitated as to stop viral
production. This is precisely what was found in 3T6 and BHK cells infected with EMC,
SFV or mengo virus (Carrasco, 1978). When we measured the uptake of [3H]GppCH2p by BHK cells, however, we found no difference between SFV-infected and
uninfected cells, despite the fact that protein synthesis was indeed inhibited more
strongly by GppCH2p in infected, than in uninfected cells. This apparent anomaly was
partially resolved when it was found, in confirmation of an earlier report (Whitehead et
al., 1981), that infected cells have a lower GTP/GDP ratio than uninfected cells, making
the GTP analogue GppCH2p a relatively more potent inhibitor of protein synthesis
(Gray et al., 1983c). Hence the greater efficacy of GppCH2p is due not to an increased
intracellular concentration of GppCH2p, but to an impaired metabolism in infected
cells. Indeed, SFV-infected BHK cells become sensitive to inhibition of protein
synthesis by GppCH2p after their rate of protein synthesis has already begun to decline
(Gray and Pasternak, 1984).
Other inhibitors of protein synthesis, such as the antibiotic gougerotin, have been
shown to have an effect similar to GppCH2p in inhibiting protein synthesis in virallyinfected cells. We have found no difference in uptake of [3H]-gougerotin by infected as
compared with uninfected BHK cells (M. A. Gray and C. A. Pasternak, unpublished
results). From this, and the other results described in this section, it must be concluded
that, whatever the reason for the shut-down of protein synthesis in virally-infected cells,
it is not the result of an increased permeability at the plasma membrane.
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579
Sugar transport
The observation that BHK cells infected with VSV, SFV (Gray et al., 1983a) or HSV
(Μ. Η. James and C. A. Pasternak, unpublished results) are able to take up 2-dGlc
2–3 times better than uninfected cells has led us to an investigation of sugar transport in
infected cells. Glucose uptake is difficult to measure (because of its rapid conversion to
most cell constitutents as well as to CO2) and analogues of glucose are therefore
routinely used instead. dGlc is taken up by the glucose transport system, and
subsequently phosphorylated by hexokinase; it is not further metabolized to an
appreciable extent, and accumulation of dGlc-6-P in cells can be used as a measure of
the rate of uptake of dGlc (Wohlheuter and Plageman, 1980). 3-O-Methyl glucose (3MeGlc) is also taken up by the glucose transport system, but it is not a substrate for
hexokinase. α-Methyl glucoside (α-MeGlc) is neither a substrate for the glucose
transport system nor for hexokinase (it enters cells by simple passive diffusion only).
Using radiolabelled dGlc, 3-MeGlc and α-MeGlc, we have obtained the results
indicated in table 1. From this it is clear that it is sugar transport, not the subsequent
metabolism, that is stimulated in infected cells. Preliminary experiments suggest that
the Vmax for transport is unaffected, and that the effect is therefore on Km. Attempts to
measure the number of Glc transporter sites by binding of [3H]-cytochalasin Β
(Baldwin and Baldwin, 1981) do not indicate an increased number of sites in infected
cells, which is in keeping with the lack of effect on Vmax.
Table 1. Effect of viral infection on sugar uptake by BHK cells.
Pooled data for BHK cells infected with SFV or HSV (unpublished results of
M. A. Gray and M. H. James).
It is somewhat surprising that infection of BHK cells by viruses such as VSV, SFV or
HSV, which in each case eventually leads to cytolysis of the cells, should induce an
increased uptake of sugar. For such an increase has to date been associated with
transformation of cells by oncogenic viruses (reviewed by Pasternak and Knox, 1979),
or with the progression of non-malignant cells to malignancy (White et al., 1981, 1983);
in these cases DNA, RNA and protein synthesis are stimulated, whereas in infected cells
the opposite is the case. Clearly an increased sugar uptake is symptomatic of some other
alteration of cell metabolism. A clue as to what this might be has come from the use of
temperature-sensitive mutants of HSV. In one such mutant (Κ), which is blocked at a
very early stage in the infectious cycle, increased sugar uptake is also blocked, whereas
in other mutants that are blocked at later stages, increased sugar uptake is unaffected.
The gene affected in mutant Κ expresses a protein that, among other things, appears to
be involved in the synthesis of stress proteins; these are proteins that are synthesized
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Pasternak
when cells are subjected to various forms of stress, such as a heat shock, viral infection,
the presence of certain chemicals, etc. The function of stress proteins is unknown: the
present results suggest that one function may be related to an increase in sugar uptake.
Neuronal excitability
If the dorsal root ganglia in the spinal cord of embryonic chicks or neonatal rats are
dissected out and cultured in the presence of inhibitors of cell division, fairly pure
preparations of single neurones can be obtained; impalement with stimulating and
recording electrodes can then be used to measure electrophysiological parameters such
as excitability (i.e. the generation of an action potential). This is the system that was
used to show the rapid and transient toxin-like effect produced by Sendai virus (Forda
et al., 1982; Pasternak and Micklem, 1983) that was referred to in a previous section.
When such neuronal cells are infected with HSV, there is no discernible change in
electrophysiological properties for some hours. About 8–10 h after infection, when
viral antigens begin to detectable, one or two changes occur (James and Mayer,
1984). With one particular strain of HSV excitability declines, apparently due to the loss
of ‘fast’ Na+ channels, as previously noted by others (Fukada and Kurata, 1981). With
another strain excitability is unaltered, but the threshold is reduced to the point that
cells begin to fire action potentials spontaneously, i.e. without any applied stimulus.
Such activity, which is depicted in figure 10, has not previously been recorded; [during
Figure 10. Spontaneous electrical activity in Herpes-infected neurones.
Rat dorsal root ganglionic neurones in culture were infected with HSV1 and electrical
recordings made as described in Forda et al. (1982). The trace shows the electrical activity of
neurones 15 h after infection; uninfected cells show no spontaneous activity.
Unpublished experiments of Μ. H. James and M. L. Mayer.
How viruses damage cells
581
the preparation of this manuscript, a preliminary communication appeared (Lima et al.,
1983) that describes essentially similar results]. Since the neurones under study are
probably sensory neurones, an intriguing possibility is raised: namely that what is being
measured in vitro is in some measure related to the pain experienced by patients
harbouring certain types of herpes virus. Clearly a more detailed study of this system,
which is another example of the effects of a viral infection on the function of cells
without killing them (figure 2), is likely to be rewarding in terms of mechanism and
therapeutic approaches pertinent to the pathophysiology of infections caused by
herpes viruses.
Conclusions
This article has described some of the alterations in plasma membrane function that are
induced by viruses. Although we have restricted ourselves to rather simple systems, it is
clear that viruses are able to damage cells without lysis; indeed in the case of the toxinlike action of certain paramyxoviruses, in the absence of an infectious cycle at All. The
idea that a viral infection can affect cell behaviour without cell death or even any
obvious cytological change has recently been raised by other groups as well (Fields,
1984) and a particularly clear-cut case has been presented for the effects of lymphocytic
choriomeningitis virus on pituitary function (Oldstone et al., 1984). This is clearly an
area for fruitful research in the future.
The proposal that some of the actions of certain viruses should be considered as
toxin-like, which has been made by others also (Smith, 1983), leads one to compare the
effects of, for example, haemolytic paramyxioviruses with those of haemolytic bacterial
toxins like S. aureus α-toxin. Each agent induces hydrophilic pores through which ions
can move so freely that the capacity of the Na+ pump to exclude Na+, CI– and H2O is
overcome, with the result that cells swell and, in the case of erythrocytes, lyse. Such a
mechanism operates in the case of complement-mediated lysis also and may do so in the
case of cell-mediated lysis (Lachmann, 1983) as well. The fact that nonerythroid cells,
which are capable of membrane repair, do not necessarily lyse when exposed to low
amounts of virus, toxin or complement, but merely undergo the transient permeability
changes described in the first part of this article, coupled with the fact that extracellular
Ca2+ is able to inhibit pore-formation by these agents, opens up a new field of
membrane research: a study of the effects of extracellular Ca2+ , as opposed to the
currently much-studied effects of intracellular Ca2+, on cellular function. Just as a study
of viral genes paved the way to a better understanding of host genes, so a study of the
toxin-like action of viruses at the cell surface may lead to a better understanding of
plasma membrane function in healthy, as well as in diseased cells.
Acknowledgements
I am grateful to many colleagues, especially Drs C. L. Bashford and K. J. Micklem, for
stimulating discussion, to Barbara Bashford and Vivienne Marvell for preparing the
figures and typescript, to Glenn Alder, Rebecca Barnes and others for technical
assistance, and to the SERC, Roche Products Ltd., the Cell Surface Research Fund and
the Mrs E. D. Norman Trust for financial support.
582
Pasternak
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