Journal of General Virology (1991), 72, 541-547.
541
Printed in Great Britain
Rotavirus infection alters N a ÷ and K ÷ homeostasis in M A - 1 0 4 cells
Jesds R. del Castillo, 1 Juan E. Ludert, 2 Aleida Sanchez, 2 Marie-Christine Ruiz, 1
Fabian Michelangeli t and Ferdinando Liprandi 2.
~Laboratorio de Fisiologia Gastrointestinal and 2Laboratorio de Biologh7 de Virus, Instituto Venezolano de
Investigaciones Cientificas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela
Infection of MA-104 cells with the OSU strain of
rotavirus induced an increase in Na ÷ and a decrease in
K ÷ intracellular concentrations, starting at 4 h postinfection. These changes were not related to an
inhibition of the Na+/K ÷ pump since ouabain-sensitive
S6Rb uptake was augmented in rotavirus-infected cells
compared to control cells, whereas the [3H]ouabain
binding and Na÷/K + ATPase activity in the cell
homogenate were unaffected. Furosemide-sensitive
S6Rb uptake (Na+/K÷/2CI- cotransport) was not
modified by the infection. Passive S6Rb efflux and ZZNa
influx were augmented in infected cells suggesting an
increase in the plasma membrane permeability. The
increase in intracellular Na ÷ concentration might be
responsible for the observed stimulation of the Na+/K +
pump. This effect was dependent upon the synthesis of
viral proteins because it was abolished by addition of
cycloheximide up to 4 h post-infection. Prevention of
the increase in intracellular Na ÷ by the use of low Na ÷containing media did not modify the pattern of protein
synthesis. This suggests that changes in intracellular
Na + and K ÷ concentrations were not related to shutoff
of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair
intestinal absorptive capacity before the appearance of
histopathological lesions.
Introduction
cellular protein synthesis. Therefore, they do not seem to
be a primary cause of the observed inhibition (Hackstadt
& Mallavia, 1982; Lacal & Carrasco, 1982; Mizzen et al.,
1987; Francoeur & Stanners, 1978; Nair, 1984).
Rotavirus forms a genus within the family Reoviridae,
which has emerged in recent years as a more important
viral aetiological agent of infantile diarrhoea in humans
and many animal species. In the host, the main target
cells are the enterocytes of the small intestine, which are
lytically infected by this agent. Most rotavirus isolates
produce a lytic infection of cultured cells; however,
persistent infections have also been observed (Carpio et
al., 1981). Although the time course of viral replication
and its effect on the synthesis of cell macromolecules
appear to vary with different rotavirus strains, shutoff of
cellular protein synthesis is generally observed (Estes &
Cohen, 1989).
In this study we report that infection by a highly
cytolytic rotavirus strain induces a change in Na ÷ and
K ÷ concentrations of infected cells. This alteration seems
to be the result of changes in the plasma membrane
permeability and not of a decrease in the Na+/K +
ATPase activity. Changes in ion concentrations do not
appear to be related to the observed shutoff of protein
synthesis.
Infection of cultured eukaryotic cells by a number of
different animal viruses has been shown to result in the
alteration of intracellular cation levels (Carrasco &
Lacal, 1983; Wagner, 1984). This appears to be a rather
general phenomenon in lytic infections by either enveloped or naked viruses. The alteration of the intracellular
concentrations of Na ÷ and K ÷ has been related to
mechanisms such as a change in the plasma membrane
permeability to these ions or to an inhibitory effect on
Na+/K + ATPase activity (Lop~z-Rivas et al., 1987; Ulug
et al., 1984; Rey et al., 1988). This effect is presumed to
play a role in the induction of morphological changes in
infected cells, such as the development of cytomegaly in
cytomegalovirus-infected cells (Nokta et al., 1988), and
to contribute to the development of c.p.e. It has been
proposed that the increase in intracellular sodium
concentration is involved in the shutoff of host cell
protein synthesis (Carrasco & Lacal, 1983). A temporal
correlation between the two phenomena has been
described in cells infected with different viruses (Garry
et al., 1979; Lacal & Carrasco, 1982; Frugulhetti &
Rebello, 1989). In other systems, changes in intracellular
Na + and K ÷ levels are detected after viral inhibition of
0000-9911 © 1991 SGM
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542
J. R. del Castillo and others
Methods
Cells and virus. African green monkey kidney cells (MA-104) were
grown in Eagle's MEM supplemented with 10~ foetal calf serum
(FCS). Virus stocks of the OSU strain of porcine rotavirus were
prepared as described (Gorziglia et al., 1985). The infectivity of virus
preparations was quantified by an immunostaining technique in
microplates (Gerna et al., 1984).
Conditions of infection. Confluent monolayers of MA-104 ceils,
washed with phosphate-bufferedsaline (PBS), were infected with virus
previously treated with trypsin (10 Ixg/ml, 1 h at 37 °C). Time of virus
addition was taken as time zero for all experiments. After 1 h
adsorption at 37 °C, cells were washed twice with PBS and replenished
with maintenance medium (MEM). An m.o.i, of 10 infectious units per
cell was used in all experiments.
lntracellular and extracellular volume determination. Intracellular
water space was determined by a modification of the method of
Kletzien et al. (1975). Ceils were washed with MEM and pulsed with 5
i~Ci/ml of 3-O-methyl-D-[3H]glucose (OMG) and 5 ~tCi/ml of [14C]inulin in MEM containing 5 mM-unlabelled OMG. Monolayers were
incubated in these conditions for 30 min to allow equilibration of OMG
across the cell and distribution of inulin in the extracellular space.
Plates were then washed with ice-cold PBS containing 0.1 mMphloretin, to inhibit equilibrated OMG efflux, air-dried and disrupted
with NaOH to extract residual radioactivity. The amount of cellassociated radioactivity was compared with radioactivity in standard
volumes of [3H]OMG and [14C]inulin solutions. Intracellular water was
estimated as the difference between total water (determined with
[3H]OMG) and extracellular water assayed by [~4C]inulin.
Intracellular ion concentrations. To measure intracellular ion contents
in control and infected conditions, MA-104 monolayers were washed
three times with a Na +- and K+-free solution, scraped off the dish,
resuspended in the same medium and centrifuged. The pellets were
resuspended in distilled water (200 ~tl) to lyse the cells completely.
Proteins were precipitated with 10~ TCA, and the contents of Na ÷ and
K + in the supernatants were determined by flame photometry. The
protein content of each sample was determined by the Coomassie blue
method (Gadd, 1981). lntracellular ion contents correspond to the
difference between the total measured ion contents and the extracellular ion contents. The latter values were calculated from extracellular
water volume (determined by inulin space) and incubation medium
concentration. Intracellular ion concentrations were calculated by
dividing the intracellular contents by the volume of intracellular water
(determined by the OMG-inulin method).
Na+/K÷ ATPase activity. The Na+/K ÷ ATPase activity was
determined in homogenates of mock- and virus-infected cells as
previously described (del Castillo & Robinson, 1985). Briefly, samples
of the homogenates were incubated with 5 mM-MgClz, 100 mM-NaCI
and 20 mM-KC1 in 50 mM-Tris-HCl, in the presence or absence of 1
mM-ouabain. The reaction was started by the addition of 5 mM-ATPTris. The liberated phosphate was measured as described by Forbush
(1983). Nat/K + ATPase activity corresponds to the fraction of the total
activity inhibited by ouabain.
Determination of S6Rb fluxes
(i) Uptake. Confluent cultures grown in 24-well plates were infected
with 0.2 ml of an appropriate dilution of virus. After 1 h adsorption
monolayers were washed twice with PBS and replenished with 0.5 ml of
MEM supplemented with 2 ~ FCS. Twenty minutes before the
addition of the label, the maintenance medium was removed and
replaced with 0.25 ml of MEM containing 25 mM-HEPES pH 7.0, with
or without drugs. Uptake at given times post-infection (p.i.) was started
by substituting the MEM-HEPES medium for a similar medium
containing S6Rb (2 p.Ci/ml). At selected times, the medium was
aspirated and the cultures were quickly washed four times with ice-cold
PBS. After drying, cells were dissolved with 0.2 ml of 1 M-NaOH.
Following neutralization with 0.05 ml of 1 M-HCI, radioactivity was
determined by scintillation counting. When appropriate, 1 mMouabain or 3 mM-furosemide was added to the incubation medium 20
min before addition of the label. Ouabain-sensitive and furosemidesensitive 86Rb uptake was calculated by subtracting the uptake in the
presence of ouabain or furosemide from the uptake in the absence of
inhibitors, respectively. Uptake in the presence of ouabain and
furosemide was considered as passive uptake. In some experiments,
86Rb uptake was also measured in a medium containing 20 mM-NaC1
(low Na t medium) prepared from individual components, using Nmethyl-glucamine (110 mM) as a Na t replacement.
(ii) Efftux. Confluent monolayers of MA-104 cells grown in 24-well
plates were mock-infected or infected with rotavirus and incubated at
37 °C with MEM supplemented with 2% FCS. At 6 h p.i., the
maintenance medium was removed and replaced with 0.2 ml/well of
MEM-HEPES medium containing 80 ~tCi/ml of 86Rb. After a 2 h
loading period at 37 °C, the radioactive medium was aspirated and the
monolayers were washed three times with PBS. Then, 200 ~tl of nonradioactive MEM-HEPES medium, containing ouabain (1 mM) and
furosemide (3 raM), were added. At selected times, 25 p.l of incubation
medium was removed for radioactivity determination. Non-radioactive medium was added to restore the original volume in the well. After
the last sampling, cells were washed with ice-cold PBS, air-dried and
disrupted with NaOH to extract residual radioactivity. Cell-associated
radioactivity at time zero was taken as 100% and results are expressed
as the percentage of radioactivity remaining inside the cell.
Passive 2ZNa uptake. Monolayers in control or infected conditions
were washed three times with low Na t medium and incubated in
MEM-HEPES medium containing Z2Na (1 ~tCi/ml). Uptake was
measured in the presence of ouabain (1 mM) and furosemide (3 mM)
to inhibit sodium movement through the Nat/K + pump and
Na+/K+/2C1 - cotransport. At given times after addition of the label,
the medium was removed, cultures were washed four times with icecold PBS and radioactivity was determined as previously described.
Uptake was linear at least during the first 3 min.
[3H]Ouabain binding. MA-104 cells were incubated in MEM-HEPES
medium containing [3H]ouabain (0.1 mM) for 10 min at 37 °C. At the
end of this period, incubation medium was aspirated, cells were washed
three times with ice-cold PBS and cell-associated radioactivity was
measured. These assays yield the so-called total binding. In order to
determine the non-specific binding, samples of ceils were exposed for
15 min to 5 mM-oubain before being incubated with the labelled
ouabain. The difference between the total binding and the non-specific
binding provides a measurement of the specific binding.
Labelling and analysis of proteins by PAGE. Confluent monolayers,
infected at an mo.i. of 10, were overlaid with methionine-free MEM.
At hourly intervals after infection the cultures were pulsed for I h with
25 ~tCi/ml of pS]methionine. At each time, cells were collected from
the flask with a rubber policeman, washed in PBS and lysed for 10 min
on ice with lysis buffer (0-01 M-Tris-HC1, 0.1 M-NaC1, 2 mM-PMSF and
1~ NP40). After pelleting of the nuclei at 2000 g for 3 min,
supernatants were collected and stored frozen for electrophoretic
analysis. SDS-PAGE on 10~ polyacrylamide gels and autoradiography were performed as previously described (Gorziglia et al., 1985).
Statistics. Differences between means were evaluated by the
unpaired Student's t-test. Differences were considered significant at
P < 0-05.
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543
1on transport in rotavirus-infected cells
Results
120
Intracellular N a + and K + homeostasis in rotavirusinfected cells
I
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~lO0
c~
The intracellular Na ÷ and K ÷ concentrations of MA-104
cells, at different times after infection with the OSU
strain of rotavirus and in mock-infected cells, are shown
in Fig. 1. An increase in Na ÷ and a decrease in K ÷
concentrations occurred in infected cells, starting at 4 h
p.i. This effect increased with time and at 8 h p.i.
intracellular Na ÷ concentration was threefold higher
than K + concentration. At this time, no gross morphological change in the infected cells was apparent nor was
viability, estimated by trypan blue or ethidium bromide
exclusion, affected.
In the normal cell, the low Na ÷ and high K +
concentrations are maintained by two major mechanisms, in addition to the membrane's intrinsic permeability to these ions. The first is the exchange of Na +
for K ÷, mediated by the Na+/K + pump, which is
specifically inhibited by ouabain. The second is the Na ÷,
K ÷, 2C1- cotransport system, which is sensitive to the
action of diuretics like furosemide. To evaluate the
relative contributions of these mechanisms to the
changes in Na ÷ and K ÷ contents in cells infected by
rotavirus, we studied the effect of ouabain and/or
furosemide on the transport of 86Rb, a substitute for K ÷.
Fig. 2 shows the time course of 86Rb uptake in control
and infected MA-104 cells, at 4 h p.i. In both groups,
86Rb uptake was linear at least during the first 2 min.
Rotavirus infection produced an increase in initial 86Rb
uptake.
In control cells, ouabain and furosemide inhibited
initial 86Rb uptake by 4 6 ~ and 33~, respectively. The
uptake obtained in the presence of both drugs (considered as the passive, non-mediated uptake) represents
about 1 9 ~ of the total uptake. Ouabain-sensitive,
furosemide-sensitive and passive 86Rb uptake at 4 h p.i.
and 8 h p.i. are shown in Fig. 3. In infected cells the
ouabain-sensitive and the passive uptakes but not the
furosemide-sensitive uptake were increased. The effect
was more marked at 8 h p.i. These results indicate that
the changes in ion concentrations were not related to
inhibition of the Na+/K ÷ pump, the activity of which
appeared to be stimulated, but rather to an increase in
the plasma membrane permeability, as suggested by the
increase in the passive 86Rb uptake.
To distinguish whether the increase in the activity of
the pump was due to an increase in the number of sites or
to stimulation of the units already present, we estimated
the activity of the Na+/K + ATPase, the biochemical
expression of the Na÷/K + pump, and estimated the
number of pumps by [3H]ouabain binding. At 8 h p.i.
neither the Na÷/K ÷ ATPase nor ouabain binding were
8O
r~
8
60
4o
20
--
~
0
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0
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2
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4
6
Time p.i. (h)
Fig. 1. Intracellular cation concentrations in MA-104 cells infected
with rotavirus. Intracellular concentrations of N a ÷ ( O , O ) and K ÷ ( A ,
A ) at different times in mock-infected cells ( O , A ) and in cells
infected at time zero with rotavirus strain O S U ( 0 , A ) . Points are
m e a n values + S.E.~I. of three experiments.
20
O
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1
2
3
Time (min)
4
5
15-
~10
x
5
0
Fig. 2. 86Rb uptake in rotavirus-infected cells. Time course of 86Rb
uptake in control ( O ) and infected cells (O), measured at 4 h p.i.
Results are m e a n values ___S.E.M. of three experiments.
affected by virus infection, indicating that the increased
ouabain-sensitive 86Rb uptake was due to a stimulation
of pre-existing pumps (Table 1).
The plasma membrane permeability to N a ÷ and K +
was evaluated by measuring the passive 22Na uptake and
86Rb efflux, respectively. As shown in Table 2 and Fig. 4
the permeability to both ions was increased in rotavirusinfected cells.
These results suggest an increase in the activity of the
pump as a compensatory response to the increased N a +
concentration. To verify this hypothesis we tested the
86Rb influx in cells maintained, after infection, in
medium with a lower Na ÷ concentration (20 mM). As
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544
J. R. del Castillo and others
100
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Fig. 4. S6Rb efltux at 8 h p.i. in rotavirus-infected (Q) and mockinfected (C)) cells. Cell-associated radioactivity at different times is
expressed as the percentage of cell-associated radioactivity at time
zero. Points are the mean values of four replicate wells.
.~ 4
5
NS
~ ~× 4 -
~.~
A
B
C
,,o
Fig. 3. Ouabain-sensitive (A), furosemide-sensitive (B)and passive(C)
influx in mock-infected (empty bars) and rotavirus-infected (hatched
bars) MA-104 cells at 4 (a) and 8 (b) h p.i. Results are mean values
+ S.E.M. of three experiments. Levels of significance of the difference
between control and infected cells, indicated in the figure, were
evaluated by Student's t-test. NS, Not s i g n i f i c a n t ; . , P < 0.05; **,
p<0.01.
~ ~.--- 2
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Normal
medium
T a b l e 1. Na+/K + ATPase activity and specific [3H]ouabain
binding in mock-infected (control) and rotavirus-infected
MA-104 cells at 8 h p.i.
ATPase activity*
(nmol/mg protein x min)
[3H]ouabain binding*
(pmol/mg protein x min)
Control
Infected
67-3 + 5-38
64.6 + 4.52
1-4 + 0.05
1.5 + 0.10
Fig. 5. Ouabain-sensitive 86Rb uptake in rotavims-infected cells
(hatched bars) and control cells (empty bars), maintained in normal
medium (130 mM-Na +) and low Na + medium (20 mM-Na+). Measurements were made at 4 h.p.i. Results are expressed as mean values
+S.E.M. of three experiments. NS, Not significant; ,, P < 0 . 0 1
compared to control.
Table 2. 22Na
influx in MA-104 cells infected by rotavirus
2ZNa influx*
(c.p.m./mg protein x rain)
* Results are mean values + $.E.M. of three determinations.
Condition
shown in Fig. 5, reduction of extracellular N a ÷ concentration from 130 mM to 20 mM decreased the total entry of
86Rb in both control and infected cells and abolished the
increase in influx caused by rotavirus infection.
Low Na ÷
medium
Control
Infected
4 h p.i.
8 h p.i.
6636 __ 208
8636 __ 150t
6595 _+ 283
11225 _+451t
* Results are mean values _+S.E.M. o f three different determinations.
t P < 0-001, compared to mock-infected condition (control).
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1on transport in rotavirus-infected cells
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Fig. 6. Timecourseof protein synthesis in MA-104cellsinfectedwith the rotavirusstrain OSU. Cells, infectedat an m.o.i, of 10, were
labelledwith [3sS]methioninefor I h intervals. Time of virus additionwas taken as time zero. Numberedintervalsindicatethe labelling
period in h p.i. ; other lanes represent (M) mock-infectedcells and caesium chloride-purifiedpreparations of single capsid (S), double
capsid (D) and empty particles (E) of rotavirus.
Synthesis of proteins in rotavirus-infected cells
Modification of the intracellular cation concentrations
by virus infection has been proposed as a possible
mechanism associated with inhibition of cellular protein
synthesis in a number of virus-cell systems (Carrasco &
Lacal, 1983). We therefore studied the kinetics of protein
synthesis under the same conditions (virus strain, m.o.i.)
in which the effect on membrane permeability had been
demonstrated. Protein synthesis in rotavirus-infected
MA-104 cells, pulsed at hourly intervals with [35S]methionine, is shown in Fig. 6. It can be seen that synthesis of
viral proteins is initially detected at the second hour p.i.,
reaches a maximal rate at the fourth hour p.i. and
declines at the eighth hour p.i. Increasing inhibition of
cellular protein synthesis occurs starting at the fourth
hour p.i. At this time the intracellular Na + concentration
is increased by approximately 20%. To study the possible
relationship between the two phenomena, protein
synthesis was studied in cells maintained in low (20 mM)
N a ÷ medium. Under this condition the increase in 86Rb
influx induced by virus infection is abolished (Fig. 5). As
shown in Fig. 7 the shutoff of cellular protein synthesis in
infected cells, measured at 5 and 6 h p.i., was not affected
by the low extracellular Na ÷ concentration.
To evaluate whether changes in plasma membrane
permeability were mediated by the synthesis of new
proteins induced by rotavirus infection, the effect of
cycloheximide, an inhibitor of protein synthesis, on
ouabain-sensitive 86Rb influx was determined (Fig. 8).
Addition of cycloheximide up to 4 h p.i. prevented the
increase in 86Rb in infected cells. The drug did not affect
the influx in uninfected cells.
Discussion
Rotavirus infection produces in both the nucleus and
cytoplasm of the host cell a variety of cytopathological
changes, which include a generalized inhibition of the
cell macromolecular synthesis (Carpio et al., 1981). In
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546
J. R. del Castillo and others
1
2
b
S
3
a
b
a
b
VP1 VP2--
VP6-
~::ii,~ii~• •!!!~i!~,~
~¸•
Fig. 7. Lack of effect of low extracellular N a ÷ concentration on protein
synthesis in rotavirus-infected cells. Control (lanes 1) and rotavirus(lanes 2 and 3) infected cells were incubated in normal (a) or low N a ÷
(b) media. Cultures were pulsed with [35S]methionine during the fifth
(lanes 2) and sixth (lanes 1 and 3) h p.i. and analysed by P A G E .
Identical amounts of protein (22 p~g/lane) were loaded for all conditions
tested. S, Single capsid O S U rotavirus,
k
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4
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//
Time of cycloheximide addition
(h.p.i,)
Fig. 8. Effect of cycloheximide on the ouabain-sensitive 86Rb influx in
rotavirus-infected cells. Cycloheximide (l 0 ~tg/ml) was added to control
( O ) and to infected (O) cells at different times p.i. and initial ouabainsensitive 86Rb influx was measured at 9 h p.i. Influx determinations in
control (/k) and rotavirus-infected ( A ) cells maintained without
cycloheximide were also measured at the same time. Points are the
m e a n values of four replicate wells.
this report, we show that rotavirus infection of MA-104
cells leads to a modification of the intracellular monovalent cation concentrations. This change might have
marked effects on the metabolism of the infected cell and
may impair some of its functions before lysis or
detachment from its substrate occurs. Alteration of
monovalent cation levels is a common result of infection
by viruses with different replication strategies including
cytoplasmic naked viruses (picornavirus), enveloped
viruses (rhabdovirus, alphavirus) and nuclear enveloped
viruses (cytomegalovirus) (Nair, 1984; Lacal & Carrasco, 1982; Francoeur & Stanners, 1978; Garry et al.,
1979; Nokta et al., 1988). Mechanisms by which
homeostasis of ions is affected appear to vary between
different viruses. In the case of rotavirus, we found that
the mechanism that most likely accounts for the change
in intracellular ion concentrations was an increase in the
passive permeability to monovalent cations rather than
to an inhibitory effect on the N a t / K + pump activity.
This conclusion is supported by the observation in
infected cells of (i) an increase in the passive 86Rb efflux
and 22Na uptake; (ii) a stimulation of the ouabainsensitive 86Rb uptake, indicating a stimulation of the
N a t / K ÷ pump; and (iii) no changes in the number of
N a t / K + pumps and in the activity of the N a t / K +
ATPase. This situation is similar to the one observed in
cells infected with picornavirus or cytomegalovirus
(Nair, 1984; Lacal & Carrasco, 1982; Nokta et al., 1988)
but completely different from the one observed after
infection with togavirus or arenavirus (Ulug et al., 1984;
Rey et al., 1988), in which ion concentration changes
appeared to be related to inhibition of the N a t / K +
ATPase activity. In addition, we have found that
rotavirus infection increases intracellular free Ca 2÷
concentration with approximately the same time course
as the changes observed in monovalent cation homeostasis (Michelangeli et al., 1991). The relationship between
the increase in Na t, K ÷ and Ca 2÷ permeability remains
to be established.
It has been proposed that changes in the intracellular
levels of Na t and K ÷ are causally related to selective
shutoff of cellular protein synthesis (Carrasco & Lacal,
1983). Our results with rotavirus-infected cells show that
a marked shutoff of cellular protein synthesis coincided
with a significant change in Na ÷ and K ÷ levels.
However, prevention of the increase in intracellular Na t
concentration by the use of low Nat-containing media
did not modify the pattern of protein synthesis. In this
condition the intracellular K ÷ concentration should
remain low due to the decrease in pump activity and the
supposed increase in permeability to ions. This suggests
that inhibition of cellular protein synthesis by rotavirus is
independent
of
altered
monovalent
cation
concentrations.
The molecular basis of the virus-induced changes in
membrane permeability, which implies the identification of the virus-encoded product responsible for the
effect, remains to be defined. Insertion of viral proteins
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Ion transport in rotavirus-infected cells
in the plasma membrane has been proposed to play a role
in the alteration of ion concentrations induced by
coronavirus or alphaviruses (Mizzen et al., 1987; Ulug et
al., 1984). One rotavirus structural protein, VP4, the
product of gene 4, appears to be able in its cleaved form
to interact with the cell plasma membrane affecting its
continuity and allowing virus penetration (Kaljot et al.,
1988). Two rotavirus proteins (VP7 and NS28, products
of genes 8 or 9 and 10, respectively) are inserted into the
endoplasmic reticulum membrane (reviewed by Estes &
Cohen, 1989), but no evidence has been reported so far
for the interaction of any rotavirus product with the
plasma membrane. Alternatively, a virus product might
affect membrane permeability through an indirect
pathway.
Defective Na ÷ transport and/or carbohydrate malabsorption, due to extensive atrophy of the villi, have been
considered as primary mechanisms in the establishment
of diarrhoea induced by rotavirus infection. However, in
some cases the onset of diarrhoea has been shown to
precede villous atrophy (Crouch & Woode, 1978; Theil et
al., 1978), suggesting functional alterations in the small
intestine before the appearance of histopathological
lesions. A mechanism responsible for these functional
changes could be an alteration of the homeostasis of Na ÷
and K ÷ in the infected enterocytes, as is found to occur in
cultured cells. The increase in plasma membrane
permeability would increase the intracellular Na ÷ concentration, reducing the Na ÷ gradient across the apical
membrane of absorptive cells and in this way decreasing
Na ÷ and sugar absorption. Thus, rotavirus-induced
impairment of intestinal absorptive capacity, preceding
massive enterocyte death, could constitute an initial
stage of the development of diarrhoea.
We wish to thank D. Otero for the drawings and J. Rivas for the
photographic work. This work was supported by CONICIT grant no.
S1-1832 and INSERM grant no. 2/489NS1.
References
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(Received 11 September 1990; Accepted 28 November 1990)
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