Archives of Virology 73, 171-183 (1982)
Disintegration of Retroviruses by Chelating
Agents
By V. Wunderlich and G. Sydow
Central Institute for Cancer Research, Robert-Rossle-Institute,
Academy of Sciences of the German Democratic,
Berlin
German Democratic Republic
With 2 figures Accepted April 2, 1982
Summary
Exposure in vitro of various mammalian
retroviruses to the chelating agents EDTA or EGTA in millimolar
concentrations resulted in partial disintegration of viral membranes
as measured by accessibility or even release of reverse
transcriptase, an internal viral protein, without any other
treatment usually required. AMong the viruses responding to
chelators were mammalian type C viruses, primate type D viruses and
Bovine leukemia virus. The effect was dose-dependant. The avain
type C virus AMV, howeveer, was found to be not susceptible to the
agents. Rauscher mouse leukemia virus treated in vitro with EDTA or
EGTA showed reducedinfectivity in mice. The results are considered
as evidence for some association of divalent cations with membranes
of mammalian retroviruses. The disintegrating activity of EGTA
suggests that Ca2+ is an integral constituent of viruses but Mg2+
may also be involved. These cations seem to be responsible for
maintaining the integrity of retroviral membranes which, after
chelation of ions, are either disrupted or become permeable for the
exogenous template of reverse transcriptase. In addition, the
disintegrating activity of trifluoperazine may indicate that a
calmodulin-like protein occurs in retroviral membranes.
Introduction
Retroviruses contain a single stranded RNA genome and the
enzyme reverse transcriptase (RT), both of which are located with
the virion core. Viral particles are released by budding from the
surface of infected cells and the cores are thought to acquire in
this process an outer unit membrane of host cell origin. However,
little is known about the composition, architecture and topography
of the viral membrane witht he possible exception of spikes and
knobs protruding from the exterior of the envelope. The
porphological elements are composed of predominantly glycosylated
proteins encoded in the viral genome and determine many of the
biological properties of the virus. (see 23 for a review).
Therefore, previous investigations on the retroviral envelope have
mainly been focused on those components.
The demonstration of RT activity requires the disruption of
virus particles as usually performed with the aid of detergents.
Our observation that the preparation of type D retroviruses in
buffers containing the chelating agent ethylene-diaminetetraacetic
acid (EDTA) results in considerable accessibility or even release of
RT activity without the use of any detergent (32) led us to
investigate more systematically the action of chelating agents on
different types of retroviruses. The results presented in this
study demonstrate that susceptibility to such agents is a
characteristic property of various retroviruses. Thus, the
structure of retroviral membranes obviously stabilized by divalent
cations may be more complex than hitherto known. Appreciation of
this complexity may allow a better understanding of certain aspects
of virus-cell interaction and may facilitate the future design of
antiretroviral compounds.
Materials and Methods
Chemicals
EDTA (Chelaplex III, p.a.) was purchased from VEB Berlin-Chemie,
Berlin-Adlershof, GDR. EGTA [ethylene glycol bis 92-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, Dr. Th. Schuchardt GmbH., Munchen,
FRG) was kindly provided by Dr. H. Will, Berlin. Propranol was
obtained from Isis-Chemie KG< Zwickau, GDR, either as solution 9 1
mg/ml) ready for injection or ina solid form. Crystalline samples
of congeners of propranol were generously furnished by the Imperial
Chemical Industries Ltd., Pharmaceutical Division, Macclesfield,
CHeshire U.K. (practolol, atenolol) and the Mead Johnson
Pharmaceutical Division, Evansville, Indiana, U.S.A., through
courtesy of Dr. K. W. Wheeler (sotalol-HCL). Trifluoperazine was
purchased from Smith Kline & French laboratories, Hertfordshire,
U.K., as a solution. The same company kindly supplied TFP in a
crystalline form.
Viruses
Avian myeloblastis virus (AMV) was isolated from the plasma of
leukemic chickens. Raucher leukemia virus (RLV) was isolated from
the plasma of RLV- infected leukemic NMRI mice or from supernatants
of the persistently infected continuous mouse cell line Y 8e. The
other viruses were propagated in tissue culture. Simian sarcoma
virus (SSV) containing an excess of simian sarcoma associated virus
(38) was grown in the human lymphocyte cell line NC 37. Bovine
leukemia virus (BLV) was isolated from chronically infected fetal
lamb kidney cells (34) provided by Dr. A. Burny, Brussels, Belgium.
Mason-Pfizer monkey virus (MPMV), originally isolated from rhesus
monkeys (8), was grown either on the human ovarial carcinoma cell
line Tu 197 (14) or the human rhabdomyosarcoma call line A 204.
Squirrel monkey retrovirus (SMRV) (15) was also propagated in A 204
cells. PMF virus (PMFV), a retrovirus first isolated from malignant
permanent human cells (14) and shown to be closely related to but
different from MPMV (33) was grown in Tu 197 or A 204 cells. SSV,
MPMV, and SMRV as well as uninfected NC 37 and A 204 cells were
originally provided by the Viral Oncology Program, Office of Program
resources and Logistics, NCI Bethesda, MD, U.S.A., through the
courtesy of Dr. J. Gruber. Virus producing cells were maintained in
suspension (A204, NC 37) or monolayer cultures (all others) in glass
flasks in either RPMI 1640 or Eagle MEM supplemented with 5-10 per
cent heat-inactivated fetal or normal calf serum and antibiotics.
Culture medium was daily replaced and cells were grown to near
confluency.
Viruses were concentrated from clarified (14,000 x g, 15
minutes) plasma or tissue culture fluid by centrifugation (50,000 x
g, 120 minutes) through a cushion of 30 percent sucrose (w/v) in TN
buffer (0.01 mol/l Tris-HCl, 0/1 mol/l NaCl, ph 8.3). Sometimes
viruses were further purified using both velocity sedimentation and
isopycnic centrifugation in sucrose density gradients (33). To keep
the level of spontaneous release of RT activity as low as possible,
freshly harvested virus and a short preparation procedure were
usually preferred but there was, in principle, no difference in the
response of semi- or highly-purified virus to the agents under
study. If necessary, virus preparations were stored at -20C until
used.
In Vitro Protocol
As described elsewhere (41), two approaches were used to
evaluate the effect of agents on retroviruses in vitro.
In intial experiments, chelator was used in place of detergent
in the preincubation step before RT assay, 20 ul of a solution of
either EDTA, EGTA (2 mmol/l) or Nonidet P40 (NP 40, 0.1 pet cent) in
0.01 mol/l Tris-HCl buffer, pH 8.3, were added to 20 ul virus
suspension (about 4 ug of protein) containing 0.1 mol/l
dithiothreitol. Incubation was performed at 0C for 30 minutes. The
RT reaction mixture (10 ul) was then completed and the assay was
done as described (40) using poly rA.oligo dT (Boehringer) as a
primer-temlate. However, the concentration of divalent cations was
raised in equivalence to the amount of previously added chelator.
In the majority of experiments, however, every virus
preparation was subdivided into four aliquots assayed each for RT
activity after treatment as follows:
(a) virus not treated with agent and incubated without detergent
(b) virus not treated with agent and incubated with NP 40.
(c) virus treated with agent as indicated and incubated without
detergent.
(d) virus treated with agent as indicated and incubated with NP
40.
Treatment of virus with agents in aliquots (c) and (d) was
carried out by resuspending high sped viral pellets thoroughly
(approximately 4 ug of protein/ml) in 5 ml of 0.01 mol/l Tris-HCl
buffer, pH 8.3, containing the agents as indicated, followed
immediately by pelleting at 50,000 x g for 60 minutes at 4 C.
Control aliquots (a) and (b) were handled in the same way under
omission of agents. The final pellets were resuspended in 100 ul
Tris buffer, incubated at 0 C for 30 minutes with or without 0.05
per cent NP 40, and assayed for RT activity as described (40) using
either Mn++ (0.3 mmol/l for RLV and SSV) or Mg++ (9.6 mmol/l, all
other viruses) as a divalent cation. Reaction mixtures were
incubated at 37 C except those of BLV and RLV having their optima at
25 and 30 C, respectively. The lytic activity (LA) of a given agent
was defined as the difference of the amount of undisrupted virus
present before and after treatment with agent referred to the
initial amount of undisrupted virus. LA was calculated by the means
of the formula:
LA= 1- (d-c) x 100
b-a
where a,b,c and d refer to the activities of RT (dis/min
incorporated into acid insoluble material) in the above mentioned
aliquots. Incorporation of radioactivity in controls (b) was in the
range of 50,000 to 200,000 dis/min. Spontaneous release of RT
activity in controls (a) was variable with different virus
preparations but did not exceed a level of 10 - 15 per cent of
controls (b), otherwise the virus was eliminated from consideration.
Proteins were determined by a micromethod based on staining
with bromophenol blue (31).
Infectivity assays with RLV
2.0 ml of a 1:50 in 0.15 mol/l NaCl (w/v) diluted cell free
extract of spleens from RLV infected leukemic NMRI mice were
incubated with 2.0 ml of a solution of EDTA or EGTA (2 mmol/l) in
0.15 mol/l NaCl or, as a control, slaine alone at 0 C for one hour.
Male NMRI mice of our inbred colony (2 - 3 months old, body weight
of about 20 g) were then innoculated i.p. with each 0.1 ml of
extract treated or not with one of the chelating agents. Animals
were kept under conventional conditions. On day 13, half of mice
were killed and mean spleen weights in the treated and control
groups, consisting each of 10 animals, were analyzed. At different
times after injection activity of particle associated RT was
determined in pooled bloods obtained by orbital bleeding from each 5
mice of treated or control groups, respectively. The remaining
mice were kept until death to ascertain mean survival time.
Results
Disintegration of Retroviruses by EDTA and EGTA as
measured in the RT release Assay
Table 1 shows the result of a representative experiment
performed to directly explore the action of chelating agents on
retroviral membranes. When adding during the preincubation
preceding the RT assay, EDTA and EGTA obviously disassociated some
membrane components as indicated by the appearance of RT activity in
otherwise undestructed virus samples. This effect however, was
sometimes hard to reproduce possible owing to some irreversible
chelator induced activation of RT, a zinc metallozyme not
reactivable by Mg++ or Mn++ (25), even although either ion was
additionally added to the RT reaction mixture in amounts equimolar
to the chelator.
(Tables not included here but an be faxed to you if you are
interested in seeing them.)
Efficacy of EDTA and EGTA in Disintegrating
Various Retroviruses
To circumvent the problems just described, most experiments
were performed in a fashion allowing a more favorable ratio of
chelator to virus during exposure as well as the subsequent
elimination of major amounts of chelating agents before assaying for
RT activity, i.e., sedimentation of viruses through a solution
containing the agent under study. Results are expressed as lytic
activity LA as defined in Materials and Methods. This index
reflects the ability of a given agent to bring about a partial or
complete disintegration of virus particles. Partially disintegrated
particles, although to a variable extent damaged by exposure to
chelator, remain sedimentable but exhibit RT activity as seein in
aliquots (c) treated with agent but not with detergent. On the
other hand, the proportion of virus particles completely
disassembled during exposure may be obtained by the difference of RT
activities appearing in aliquots (b) and (d) treated without or with
agent followed by disruption of the remaining virus with detergent.
To give an example with RLV exposed in aliquots (c) and (d) to 1
mmol/l ETA: the four viral aliquots displayed RT activities of (a)
1.1, (b) 38.0, (c) 10.4, (d) 32.7 dis/min (each x 10 to the third),
respectively, yielding an LA value of 40. It appeared, however, not
meaningful to discriminate between partial and complete
disintegration and the LA value included by definition both events
was therefore used.
Fig. 1 (not shown) depicts the action of EDTA and EGTA on
various retroviruses. These include mammalian type C viruses (RLV,
SSV), primate type D viruses (MPMV, SMRV, PMFV) and BLV. Both
chelating agents exert in millimolar concentrations a pronounced
lytic activity upon most of the viruses tested so far. Mammalian
type C viruses seem to be somewhat less susceptible than the other
viruses. The avian type C virus AMV, however, was exceptional in
that it did respond to neither EDTA or EGTA. The reason for that
was not investigated but may be related to some peculiarities of the
envelopes of the avian type C viruses as compared to their mammalian
counterparts (23). Since RLV just as AMV was obtained from the
plasma of leukemic animals and there was no difference in the
susceptibility of RLV irrespective of originating from infected
animals or tissue cultures, it appears to be unlikely that
unresponsiveness of AMV is due to certain conditions of
extracellular viral maturation.
Concentration Dependence of Disintegrating
Activity of EDTA and EGTA
In initial experiments it was noted that retroviruses respond
to chelating agents in a dose dependant manner. Fig. 2 (not shown)
illustrates that PMFV, studied in greater detail, is susceptible to
chelators over a wide range of concentrations. Complete
disintegration, however, was reached with neither EDTA nor EGTA even
in high concentrations up to 50 mmol/l. This may indicate that only
a certain fraction of particles present in a given virus population
is sensitive to these agent possible owing to variations in age,
stage of maturity, or other factors. Such variables are known to
influence the response of retroviruses to detergents (43).
Which Ion Is Involved in Disintegration
A major question arising from the experiments described so far
is the nature of the cation complexed by the chelating agents and
probably somehow responsible for the integrity of viral particles.
Effectiveness of EGTA with its high binding affinity for Ca++
(stability constant log K=10.9) and relatively low affinity for Mg++
(log K=5.9) (3) clearly supports a role of calcium in virus
integrity. However, the ability of EGTA to produce disintegration
even in low concentrations may suggest that magnesium is also
involved in maintaining the integrity of retroviruses, since the
EDTA has binding affinities to both Ca++ (log K = 10.7) and Mg++
(log K = 8.9). The low amount of virus available for analysis did
not yet allow an identification of the respective cation(s) by means
of chemical or physicochemical methods.
The possibility that EDTA and EGTA chelate different cations in
retroviral membranes led us to examine a possible synergistic action
of both agents on retroviruses. Thus far, however, no increase in
LA values has been observed after exposure of viruses to equimolar
mixtures of both chelators.
To further substantiate the involvement of one of the ions
under consideration, experiments were performed to ascertain whether
the addition of divalent cations could prevent the action of EDTA or
EGTA (not shown). As already reported for PMFV (32) and now
confirmed with other viruses, both MgCl2 and CaCl2 prevented
disintegration of retroviruses when simultaneously added with the
chelating agent. However, addition of these cations at a later time
did not cancel the effect produced by EDTA or EGTA. These results
corroborate the assumption that both Ca++ and Mg++ ions are
associated with retroviruses. Moreover, they exclude the
possibility of involvement of such heavy metal ions binding more
strongly to EGTA than Ca++, because in that case addition of CaCL2
or MgCl2 would not have prevented disintegration.
Effect of EDTA and EGTA on Infectivity of RLV
That chelating agents indeed adversely affect retroviral
membranes was independently demonstrated in another set of
experiments. RLV, contained in cell-free spleen homogenates, was
incubated in vitro with EDTA or EGTA and then injected into mice for
analysis of leukemogenic capacity. As compared to saline incubated
virus in the controls, this treatment led to a marked inhibition of
splenomegaly as well as to a doubling of mean survival time of
infected animals (Table 2 not shown). Therefore, disintegration of
virus particles as biochemically measured is paralleled by an
equivalent loss of infectivity. The remaining virus, however, was
still able to cause leukemia being diagnosed (courtesy of Prof. F.
Fey) at the time of death of animals. Nevertheless, antiviral
activity of chelating agents was also reflected in the pronounced
depression ofparticle bound RT activity in the plasma of mice
inoculated with treated virus. During the course of development of
leukemias, there was a considerable delay in reaching comparable
levels of RT activity in blood of treated as against control mice.
Susceptibility of BLV to Different Beta Blockers
The hypothesis that Ca++ may be associated with retroviruses
was further tested in animals with beta receptor blocking drugs.
Propranolol, a nonselective beta blocker used in medical practice,
has been shown to be able to interact with membranes under
concomitant Ca++ displacemant (2). It also disrupts membranes of
type C and type D retroviruses (41). Contrary to propranolol, some
of its congeners cardioselective beta blockers like practolol,
sotalol and atenolol lacking the hydrophobicity of propranolol, do
not essentially influence membrane phospholipids and membrane boud
Ca++ (2,17,24). For that reason it appeared of interest to examine
the susceptibility of a retroviruses to these drugs. BLV was chosen
because it was not included in our earlier studies. Table 3 (not
shown) shows that exposure to propranolol in vitro clearly
disintegrates BLV, as previously demonstrated with other
retroviruses. Exposure to one of the propranolol congeners however
produces only a small, if any, disintegration of BLV. These results
may thus provide additional evidence for calcium as the ion to take
into consideration.
Lytic Action of Trifluoperazine on Retroviruses
The effect of trifluoperazine (TFP) on retroviruses ws
investigated because (i) other phenothiazines have been found to
exert a lytic effect on retroviruses in vitro (41), (ii) drugs of
this type are known to produce, along with a variety of other
effects on biological membranes, a displacement of Ca++ from
membrane components (29) and (iii) TFP is able to bind in a Ca++
dependant manner (18,19) highly effectively to calmodulin, a
widespread regulatory protein (16), and is therefore widely
considered as a specific probe for calmodulin. In view of these
properties disintegrating activity of TFP on retroviruses could
serve as an indicator both for the presence of Ca++ and for the
identification of a putative Ca++ binding molecule in retroviral
membranes. In fact, following exposure to TFP retroviruses of type
C or type D as well as BLV displayed a release of RTactivity (table
4) [not shown]. The effect was again dose dependant. To favor a
Ca++ specific binding of TFP to viral components, exposure was
performed at pH 7.2 and not at pH 8.3 as usually, because pH values
above 7.5 diminish the specificity of binding (36) . Despite some
variations with different viruses, TFP showed a similar
disintegrating activity as various phenothiazines (41). Although
AMV failed to respond to EDTA and EGTA, this virus was found to be
susceptible to TFP, similarly as to other phenothiazines (41), too.
From the results of this set of experiments it is tempting to
conclude that retroviral membranes donot only contain Ca++ but
possible also a Ca++ binding protein (7) which may be calmodulin
like in sharing with this protein the property of responding to TFP.
Discussion
In the present work designed to evaluate the action of the
action of chelation agents on retroviruses in vitro we have mainly
used a procedure sonsisting of centrifugation of intact virus
particles through a solution of each respective agent and subsequent
assay for RT to detect disintegration of the viral membrane. The
results show that these agents are capable of disintegrating all of
the mammalian retroviruses tested. This finding was supported by
experiments showing decreased infectivity of RLV after the virus had
been treated with chelating agents.
Principally, disintegration of retroviruses in vitro may occur
either spontaneously or by treatment with membrane active agents.
Spontaneous disintegration thought to be due simply to aging of
particles is variable but usually low. On the other hand, a variety
of agents including detergents (21) , lipid solvents (21), and
neurotropic drugs (41) as well as proteins such as human (37) or
nonhuman primate complement (30) and melittin (12) cause a complete
or nearly complete lysis of retroviruses. Complement mediated
virolysis affects the P15(E) protein known to be embedded directly
into the retroviral membrane (1). Other interesting agents are the
polyene antibiotics and membrane channel formers filipin (22) and
nystatin (12) which induce some alterations in but nor
disintegration of the membranes of retroviruses while retaining
their infectivity. This study revealed still another type of
response with some dissociaton of membrane components that affects
the infectivity of virus but does not necessarily result in complete
destruction of viral particles because a variable proportion of them
remains sedimentable even after treatment with chelators. Thus,
retroviruses may exhibit a broad range of response to exogenous
factors. However, among such factors, chelators like EGTA are rather
exceptional in that they do not represent true membrane active
agents, although several observations point to a role of Ca++ in
membrane stability. (6).
There is increasing evidence for some association of Ca++ with
viruses and a role of this ion in maintaining viral structure. The
observation of calcium binding sites is nearly 20 plant viruses
including tobacco mosaic virus, and also bioenergetic
considerations, led DURHAM (10,11) to propose that Ca++ might
generally control disassembly of such viruses. Other authors
succeeded in disassociated of polyoma virus by chelation of Ca++ (4)
and reassembly of infectious viral particles by subsequent addition
of Ca++ (5). Exposure of rotavirus particles to calcium chelators
resulted in an unmasking of internal RNA polymerase activity (9).
On the basis of these findings it is tempting to assume that
association with Ca++ is a widespread property among viruses of
different families.
The viral components associated with Ca++ have not been
identified. One possibility is that the attachment of Ca++ to
phosphatidylserine (13) known to occur in the so far analyzed
retroviral membranes (28). Distinctive features of model membranes
have been attributed to interactions of Ca++ with membrane
phospholipids and there is evidence that the viral membranes behave
in this respect similarly as model membranes, e.g., in virus induced
cell fusion processes (27). On the other hand, certain proteins
could serve as receptors for Ca++ owing to their ability to bind
Ca++ in a selective and reversible fashion. The prototype of such
Ca++ binding proteins is calmodulin, which, upon binding of Ca++,
undergoes a confirmational change needed for its biological activity
(16). In the presence of Ca++, calmodulin avidly binds
phenothiazines (with TFP being the most effective one) and becomes
thereafter biologically inactive (36). Propranolol is another
antagonist of calmodulin (35). Consequently the strong retrovirus
disintegrating activity of TFP, other phenothiazines, and
propranolol (41) may be considered as preliminary evidence for the
occurrence of calmodulin like proteins in retroviral membranes,
though it remains to be identified. During complex formation of
Ca++ both with phospholipids (26) and proteins (39) there is some
synergism with Mg++ and it is, therefore, well conceivable that both
cations simultaneously occur in rteroviral membranes.
Whatever the mode of Ca++ binding to viral components is, and
irrespective of whether Ca++ is accidentally or even specifically
complexed to them, the occurance of Ca++ in retroviral membranes may
have biological implication with regard to the assembly and
disassemble of viral particles in and their budding from infected
cells. Generally Ca++ has been found to influence a wide variety of
functional properties of biological membranes.
Finally, identification of Ca++ binding viral components may
eventually prove useful in the search for new and effective
retroviral agents. The presently limited success of virus
chemotherapy with chelating agents (20) might be generally augmented
by considering such components as drug targets, too. Our recent
demonstration that haloperidol, a colmodulin binding butyrophenone
(19), exerts an antiviral effect on Raucher murine leukemia virus in
vivo (42), may support the feasibility of this approach.
References
References and tables are available by calling us. We will fax them
to you.
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