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Cytomegalovirus infectivity: Analysis of the phenomenon of centrifugal enhancement of infectivity
72, 235-243
VIROLOGY
(1976)
Cytomegalovirus Infectivity: Analysis of the Phenomenon Centrifugal Enhancement of Infectivity J. B. HUDSON,’ Department
V. MISRA,
of Microbiology,
AND
T. R. MOSMANN
University of British Columbia, British Columbia, Canada Accepted
February
of
Vancouver
V6T
1 W5,
6,1976
The phenomenon of centrifugal enhancement of infectivity was examined in some detial for the murine cytomegalovirus (MCV). Centrifugal enhancement (meaning that application of virus to monolayer cultures with the aid of a centrifugal field resulted in a ZO- to 80-fold increase in infectious centers), was observed in mouse embryo cultures from three different strains of mice, in 3T3 cells, and in rat kidney cells. Three different strains of MCV and 20 plaque-purified strains of MCV all showed the property, as did the one strain of human cytomegalovirus examined. In contrast, herpes simplex virus type 1 and type 2 did not. Centrifugal enhancement could not be explained by increased penetration of MCV or its DNA into cells and their nuclei. Other experiments involving PFU-dose response, uv-inactivation kinetics, electron microscopy, and a variety of labeling regimes with thymidine and uridine isotopes, ruled out the presence of interfering microorganisms. It is concluded, therefore, that the property of centrifugal enhancement is an inherent property of MCV particles, and furthermore, it is suggested that some cytomegaloviruses may have a tendency to enter into a nonreplicating state in homologous fibroblasts.
tures “uncharacteristic” of cytomegaloviruses, such as an unusually large genome (Mosmann and Hudson, 19731, the preponderance of multicapsid virions (Hudson et al., accompanying manuscript), and the reversible attenuation of infectivity following alternate in uivo and in vitro propagation (Osborn and Walker, 1971). Thus, the phenomenon of centrifugal enhancement of infectivity could be just another unusual feature of MCV. We have investigated the phenomenon of centrifugal enhancement in more detail, and extended the study to the human cytomegalovirus. Herpes simplex was used as a control virus not displaying the property. Although the mechanism of the effect is still not understood, the present results indicate that the property (i) is inherent in all MCV particles; (ii) cannot be explained by the presence of an interfering agent, and (iii) is probably also a property of at least one strain of human cytomegalovirus.
INTRODUCTION
Cytomegaloviruses (salivary gland viruses) are considered to be members of the herpes virus genus, although they are often grouped separately from other herpes viruses because of certain characteristic biological features (Plummer, 1973). For example, the cytomegaloviruses generally replicate relatively slowly in cell cultures and yield relatively few infectious particles when assayed by conventional techniques. In order to overcome the latter problem, Osborn and Walker (1968) applied murine cytomegalovirus (MCV) preparations to monolayer cultures with the aid of a centrifugal field, and found an increase of 20- to 80-fold in the number of infectious particles. No such effect was shown by herpes simplex or pseudorabies viruses. Other studies, however, have revealed that MCV possesses certain feaI Author dressed.
to whom
reprint
requests
should
be ad235
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
236
HUDSON, MATERIALS
AND
MISRA
METHODS
Cells Mouse embryo cultures were prepared by standard methods, and used after one to three passages in vitro. Mouse 3T3 cells and H.ep.2 cells were obtained from Flow Laboratories, human WI-38 cells from Microcan Research (Calgary), the rat NRK line from Dr. V. Klement (University of Southern California), and human skin fibroblast cells from Dr. H. Stich (U.B.C. Cancer Research Unit). All cultures were propagated in Dulbecco’s modified MEM supplemented with 20 pg/ml of gentamitin, 0.25 pglml of fungizone, and 5 or 10% fetal calf serum, in a well-humidified atmosphere containing 5% CO, at 37”. Viruses
The murine cytomegalovirus (MCV) strain used in most of the studies was the Smith strain, obtained from the American Type Culture Collection. Fresh stocks of the virus were prepared every 6 to 9 months by low multiplicity passage (co.01 PFU/cell) in Swiss mouse embryo cultures. Large quantities of virus were readily obtained by inoculating stock virus (at less than 0.01 PFU/cell) into roller bottles of mouse embryo cultures. The “virulent” strain of MCV (designated K181, from Dr. June Osborn, University of Wisconsin) was passaged twice in mouse embryo cultures to yield enough virus for examination. The Apodemus sylvaticus (field mouse) strain of MCV, obtained from Dr. Cordon Plummer (Loyola University), was treated in the same way. Herpes simplex virus (HSV) was the type 1, P strain, described previously (Mosmann and Hudson, 1973). The MS strain of HSV-2 was obtained from the American Type Culture Collection. These were passaged in H.ep.2 cells or mouse embryo cultures. The AD169 strain of human cytomegalovirus was obtained from the American Type Culture Collection, and was propagated in WI-38 cells. Viruses were purified by the procedure involving differential centrifugation and nuclease treatment, as
AND
MOSMANN
described 1973).
before (Mosmann
and Hudson,
Plaque purification of MCV. MCV (Smith strain) was purified by repeated centrifugal infection or repeated standard infection and isolation of well-separated plaques. In order to provide sufficient virus for study, the third passage involved low multiplicity infection (less than 0.001 PFU/cell) without an overlay medium, and the cell-free fluids were collected when most of the cells had become infected (3 to 5 days after infection). Plaque assay. MCV was titrated by either the centrifugal or the standard method of inoculation (Osborn and Walker, 1968). One or two milliliters of virus in MEM + 2% serum (for 35- or 50mm diameter plastic dishes, respectively) were added to drain, freshly confluent, mouse embryo monolayers. The dishes were then either centrifuged at 800g for 30 min in the International CS centrifuge, or allowed to stand at ambient temperature for 30 min. The inocula were removed and replaced with either 2 ml per 35-mm dish or 4 ml per 60-mm dish, of overlay, which consisted of MEM plus 0.5% agarose, 5% fetal calf serum, 20 pg/ml of gentamicin, and occasionally 50 units per ml of mycostatin. Plaque counts of the order of 500 to 5,000 were enumerated under the microscope 36-48 hr after infection, and less than 500 per 35-mm dish were counted macroscopically, without staining, after 3 to 6 days. Herpes simplex and human cytomegalovirus were assayed in an analogous fashion, on appropriate cell monolayers. Cell Fractionation
Monolayers of uninfected, MCV-infected, or HSV-infected, cells were washed twice with ice-cold RSB+ (0.1 M NaCl, 0.01 M Tris-chloride, pH 7.4, 0.015 M MgCl&, then with RSB (0.01 M NaCl, 0.01 it4 Trischloride, pH 7.4, 0.0015 M MgCl,). The cells were allowed to swell for 15 min in RSB, scraped off the dish, and homogenized in a Dounce homogenizer to disrupt the cells. This last process was always monitored by phase contrast microscopy to ensure complete disruption of cells with preservation of nuclear integrity. In some
CYTOMEGALOVIRUS
experiments, the washed monolayers were exposed to RSB containing 1% NP40 (Nonidet P40) for 15 min at 4”. The lysates were than shaken from the culture dishes. Nuclei were sedimented at 1000 g for 10 min and washed once. The supernatant from the first centrifugation was used as cytoplasmic extract. Control experiments with both uninfected and MCV-infected cells, containing prelabeled cellular DNA (13Hlthymidine), showed 1% or less of this label in the final cytoplasmic extract obtained by either technique, indicating preservation of nuclei. Phase contrast microscopy revealed little or no obvious contamination of nuclei by cytoplasmic fragments. Ultraviolet
Irradiation
This was samples of distance of germicidal
accomplished by placing l.O-ml virus in 50-mm dishes, at a 20 cm from a Sylvania 15 W lamp.
Nucleic
of Virus
Acid Extraction
Nucleic acid was extracted from virus samples by the pronase-SDS technique described in detail elsewhere (Mosmann and Hudson, 1973). RESULTS
Comparison of Centrifugal Infectivity
and Standard
In the presentation of these results, the term “centrifugal-PFU” refers to plaques obtained following centrifugation of the monolayers in the presence of virus, whereas “standard-PFU” denotes omission of a centrifugal field. Details of the techniques are described in the Materials and Methods section. Initially we repeated many of the experiments reported by Osborn and Walker (1968), using our tissue-culture passaged MCV, and obtained essentially identical results. Over a period of about 3 years, with many different virus preparations, the ratio of centrifugal PFU to standard PFU has varied randomly between extremes of 15 and 85, most values falling within the 25-50 range. This value is referred to as centrifugal enhancement or c/s
231
INFECTIVITY
ratio. Plaque morphology is identical in the two modes of infection comprising characteristic-looking rounded cells retracting from their neighbors and leaving a central clear hole in the monolayer which can be visualized easily without straining. The rate of growth of the plaques is similar, although in older mouse embryo cultures, the initiation of a standard plaque may lag by 24 hr. Virus which did not initially adsorb to the cells following centrifugal or standard inoculation, still displayed centrifugal enhancement upon infection of a second set of cultures. This result argues against the possibility of selective uptake of function” ally different virions, i.e., selective uptake of virions giving either standard or centrifugal modes of infection. Centrifugation of the virus or cultures separately before their mixing did not allow maximum expression of infectivity. The simultaneous presence of both components was required, although centrifugation after standard inoculation did increase plaque numbers. Centrifugal Enhancement Cell Types
in
Different
Most of the experiments described utilized embryo cultures from random bred Swiss mice, the cultures being used after one to three passages in vitro with similar results. Centrifugal enhancement was not unique to these cells, however, since inbred Balb/c and ICR embryo cultures also yielded centrifugal enhancement, as shown in Table 1. Furthermore 3T3 cells and the rat kidney cell line NRK gave the same results (Table 2). It should be pointed out that because the c/s ratio varies randomly from one experiment to another (presumably due to “physiological” variables), strict comparison between virus preparations or cell cultures, can only be made by simultaneous infections. Absolute titers and cls ratios can vary to some extent if a virus preparation is assayed over a period of several days on the same set of cultures. Although 3T3 cells gave a similar virus titer to embryo cultures, they were not used for routine virus assays
238
HUDSON, TABLE
MISRA
1
Other Viruses
MCV INFECTIVITY FROM
IN MOUSE EMBRYO CULTURES DIFFERENT STRAINS OF MOUSE
Embryonic fibroblasts from Swiss (randombred) Balblc ICR
PFU/ml (centrifugal) 2.86 x 106
22.3
2
STRAIN) INFECTIVITY CELL TYPES
Cell line ME 3T3 NRK (normal rat kidney line
42.3 35.8
PFU to standard PFU. TABLE
MCV (SMITH
c/s ration
3.60 x lo6 3.04 x 106
(1Ratio centrifugal
PFU/ml (centrifugal) 2.25 x 108 6.75 x 10’ 3.25 x IO’
IN DIFFERENT
Ratio:c/s 18.5 18.0 26.0
since they had a very narrow range of growth over which plaques were visible. Thus, 3T3 cultures 1 day past confluency no longer yielded discernible plaques. The rat kidney cells gave smaller MCV titers, and the plaques took longer to develop, which may reflect insufficient adaptation to the rat cells. Therefore mouse embryo cells were used routinely for plaque assays. Plaque-Purified
AND MOSMANN
The experiments so far were performed with the Smith strain of MCV. Table 4 shows that the Apodemus isolate of MCV also displayed centrifugal enhancement. In contrast, the K181 strain of MCV initially showed a low c/s ratio, similar to HSV-1 and HSV-2. This is interesting because the K181 strain is more virulent than the Smith strain toward mice (June Osborn, personal communication). However, after four passages in mouse embryo cultures, the c/s ratio of K181 rose to 18.0. Herpes simplex c/s ratios were always less than 8.0, and usually less than 5.0 (Table 51, these values being explicable in terms of a simple increased penetration into the cells under a centrifugal field (see below). Human cytomegalovirus (HCV, strain AD169) also showed centrifugal enhancement when assayed in human fibroblasts. These results are summarized in Table 6. In three out of the four tests centrifugal enhancement was evident, whereas HSV-1 TABLE INFECTIVITY
PFU/ ml (x 10-B
Twenty plaque-purified strains of MCV were derived, 10 by repeated centrifugal infection and 10 by repeated standard infection. Table 3 shows that all 20 strains displayed the property of centrifugal enhancement, the c/s ratios ranging from 18 to 70, with mean values of 48 and 35, respectively, for centrifugaland standardderived strains. There was no obvious difference between the two strain categories. Two strains were assayed again later and, although both showed centrifugal enhancement, the values were different, illustrating the apparent random nature of the c/s ratio. It was therefore concluded that the property of centrifugal enhancement was not attributable to genetic heterogeneity in the virus population.
tio
ard strains
20.3 48.3 18.1 43.5 37.0 28.0 60.0 43.8 47.0 48.8 39.5
Sl s2 s3 s4 s5 S6 s7 S8 s9 SlO mean
TABLE VIRUS
I
OF PLAQUE-PURIFIED MCV ,:is Ra- StandPFU/ cls
1.42 1.50 0.32 1.63 1.35 1.40 0.72 0.70 0.40 0.34
MCV
3
INFECTIVITY
Virus MCV (Smith) MCV (Apodemus) MCV (Osborn-K181) HSV-1 (Strain P) HSV-2 (Strain MS)
4
IN MOUSE
ml (x 10-S)
ratio
0.60 0.18 1.41 3.25 1.49 1.38 2.07 3.08 2.88 2.92
52.1 70.0 46.5 38.4 42.4 39.0 18.8 48.9 38.4 66.4 46.1
EMBRYO
CELLS
Ratio: Centrifugal PFU/standard PFU (c/s) 18.0 23.6 5.2 3.7 5.6
CYTOMEGALOVIRUS TABLE HSV Cell
line
ME
PFU/ml (centrifugal)
HSV-1 HSV-2 HSV-1 HSV-2
NRK
3.8 2.0 2.0 2.2
TABLE INFECTIVITY
OF HCV
Test 1 2 3 4 5
Cell
TABLE
5
INFECTIVITY
Virus
type
WI-38 WI-38 Human skin iibroblasts Human skin fibroblasts Human skin flbroblasts
x x x x
lo5 104 10” 10”
Ratio: C/S
4.2 3.9 2.9 5.6
7
ENHANCEMENT OF [3H]TdR-LanELEn VIRUS UP BY THE NUCLEUS BY CENTRIFUGAL INO~ULATIO@ Fraction
Centrifugal ‘T;;’
IN HUMAN
CELLS
Virus
c/s ratio
HCV HCV HCV
25.0 25.0 7.5
HCV
30.0
HSV-1
3.7
of MCV into Cells and Nuclei
One obvious explanation for centrifugal enhancement would be a greatly increased uptake of virus into the cells, or into the nuclei. This was initially considered plausible in view of the predominant multiple capsid virions in MCV populations (Hudson et al., accompanying manuscript). In order to test this hypothesis, MCV was labeled with tritiated thymidine. Herpes simplex type 1 was similarly labeled and used for comparison. The results of one experiment are displayed in Table 7, from which it can be seen that penetration into cells and into their nuclei was increased two- to threefold by centrifugation, for both viruses. The same result was obtained for 3T3 cells, using both of the methods of cell fractionation described in the Materials and Methods section. Thus the infectivity of MCV is increased by an additional order of magnitude by applying
TAKEN
Enhancement
Nuclear fraction
1946 2636
) 2.6-fold i 3.2-fold
Cytoplasmic fraction
1810 4752
I 1 2.8-fold i 2.5-fold
6
AND HSV
in these cells gave a ratio of only 3.7. The HCV plaques developed slowly, being invisible to the naked eye until about 9 days after infection, but the c/s ratio remained constant from their first appearance under the microscope (4-5 days p.i.) until degeneration of the monolayers set in after 3 weeks. Penetration
239
INFECTIVITY
o Monolayers of mouse embryo cells infected with l”H]TdR-labeled MCV or HSV by either the standard or centrifugal method were washed twice with ice-cold RSB+, then with RSB. The cells were allowed to swell for 15 min at 4” in RSB, containing 0.1% NP40, scraped off the dish, and homogenized in a Dounce homogenizer to disrupt the cells. Nuclei were sedimented at 1000 g for 10 min, and washed once with RSB. The supernatant from the first centrifugation was used as cytoplasmic fraction.
a centrifugal field, over the amount accounted for in terms of increased physical uptake of virus particles. The next series of experiments was designed to investigate the possibility of an interfering agent, virus or some other particle, which might normally suppress MCV replication except under centrifugal conditions. Proportionality and Virus
of Plaque-Forming Dose
Units
MCV produces very small though distinct plaques, and as a consequence, it is possible to count between 1.0 and 5000 plaques on a 35mm dish, up to 500 with the naked eye, and more than this under a microscope. This feature enabled us to examine c/s ratios over four orders of magnitude. Figure 1 shows the results for one such test, from which it is evident that the ratio, in this case 22.5, was essentially constant over this range, the dose response being linear for both centrifugal and standard infections. Ultraviolet
Irradiation
of MCV
Other enveloped animal viruses often displayed complex kinetics of uv-inactivation (e.g., Ross et al., 1971). Likewise MCV
240
HUDSON,
MISRA
AND
MOSMANN
by incubating infected cells at various times with 13Hluridine, and purifying MCV from the cell free fluids by the norwhich involved both mal procedure, DNAse and RNAse treatment. The results, in Table 8, show that relatively little
io4-
103it 5 z f B L IOZ-
IO-
I 10-7
I 10-b Virus Concentration
FIG. 1. Plaque stock of MCV was duplicate by both niques, on 50-mm
I 10-s (Relative
I lo-4 ]
number versus MCV dilution. A serially diluted and assayed in centrifugal and standard techdishes of mouse embryo cells.
does, as illustrated in Fig. 2. Both centrifugal and standard PFU decayed at similar rates, such that the c/s ratio at all uv doses was greater than 20, although the actual value varied randomly among the samples tested. We do not know if multipleand single-capsid virions have the same uv sensitivity; nevertheless, the range of uv doses employed was enough to kill all infectious entities, so that if any interfering particles were present, then they must have had the same uv sensitivity as MCV. Attempts to Label an Interfering Agent Virions labeled with [3H]- or [14C]thymidine, or 32P, and purified by differential centrifugation and DNAse treatment, always revealed a single species of DNA in sedimentation velocity or equilibrium density gradients (Mosmann and Hudson, 1973). This argues against the possibility of an interfering DNA-containing agent. However, virus purified by our normal procedure still retains the property of centrifugal enhancement. An RNA containing agent was sought
t 1.0
0.5
I I.5
I I 2.0 2.5 UV Dose Imins.)
I 3.0
I 3.5
t 4.0
FIG. 2. Effect of uv irradiation upon MCV infectivity. A stock of MCV (in MEM) was exposed to uv irradiation, and samples were withdrawn at intervals for plaque assay by centrifugal and standard techniques. The B-min samples contained zero PFU. 0, Centrifugal PFU; 0, standard PFU. TABLE INFECTED
Sample
8
OF [$H]TdR
INCOIWRATION
MOUSE
Labeling time (hr p.i.1
EMBRYO
Harvest t$e p.ir)
A. B. C. C.
TdR UR UR UR
label label label label
16-40 1-16 1-16 16-40
13H]UR
AND
40 16 40 40
BY MCV-
CELLS”
cpm per culture Cell-free supernatant
Ensymetreated virus pellet
109,570 7,800 39,650 1 55,250
81,650 <20 14,220 17,500
0 A series of mouse embryo cultures (in 50-mm dishes) were infected with MCV, and at the appropriate times, 5 @Z!i of 13HJTdR or 13HlUR were added per dish. At the indicated times, groups of four dishes were harvested for virus purification.
CYTOMEGALOVIRUS
uridine label and no enzyme-resistant label could be detected in cell free fluids harvested 16-hr postinfection (i.e., before progeny MCV is excreted), but significant amounts were found after this time, when thymidine-labeled MCV is also detected. The labeled viral pellets were examined further by sedimentation velocity in sucrose gradients. Figure 3 shows that most of the labeled material sedimented into the 60% sucrose cushion for samples A, B, and C, although some low molecular weight thymidine label was also evident. There was no significant amount of slower sedimenting particulate material, and in other experiments with thymidineand uridinelabeled MCV preparations, there were no particles that sedimented faster than MCV. Similar preparations were also centrifuged in 20-60% equilibrium sucrose gra-
Fraction
No.
3. Sedimentation velocity of [3H]TdRand 13HlURlabeled MCV. The virus specimens, described in the legend to Table 8, were centrifuged in 5-208 sucrose gradients (w/v, in PBS, 4.5 ml per tube) with a cushion of 0.5 ml of 60% sucrose. Centrifugation was carried out for 30 min at 15,000 rpm in the SW 50.1 rotor at 4”. Fractions were collected directly onto fiberglass filters for radioactive measurement. None of the “pellets” in the bottoms of the tubes contained detectable radioactivity. 0, 13HlTdR-labeled MCV; labeled 16-40 hr p.i. (Sample A); n , [3HlUR-labeled MCV; labeled 1-16 hr p.i. (Sample B); 0, [3H]UR-labeled MCV; labeled 16-40 hr p.i. (Sample C). FIG.
241
INFECTIVITY
dients. MCV showed a broad range of buoyant densities in such gradients, in contrast to the narrow band found for 13H]UR-labeled murine sarcoma virus (Kirsten strain, derived from transformed NRK cells). This heterogeneity in MCV density presumably reflects the presence of multiple-capsid virions (Hudson et al., accompanying manuscript). Nevertheless, uridine- and thymidine-labeled MCV profiles were essentially coincident, implying that the two isotopes were incorporated into the same particles. In order to investigate the nature of the uridine label in MCV, nucleic acids were extracted from the same preparations used for Fig. 3, and centrifuged to equilibrium in CsCl gradients. Figure 4 shows that uridine and thymidine labels are coincident, and band at the density expected for MCV DNA (Mosmann and Hudson, 1973). Thus the uridine label is an integral part of the MCV DNA, which could indicate either the presence of RNA bound to or covalently linked to the DNA, or that the uridine had been converted to a deoxyribonucleotide prior to incorporation into progeny DNA. The latter alternative is supported by the observation that the uridine label found in MCV DNA is resistant to pancreatic RNAse and alkaline hydrolysis both in the native and denatured DNA (unpublished results). Although more evidence is required to clarify this point, in the context of the present study it seems justifiable to conclude that our MCV prep arations do not contain significant numbers of any other RNA- or DNA-containing particles. This conclusion is reinforced by our inability to find particles other than typical herpeslike particles in thin sections of MCV-infected cells viewed in the electron microscope. We therefore conclude from these studies that centrifugal enhancement of infectivity is an inherent property of MCV particles. DISCUSSION
The results described in this communication clearly indicate that the property of centrifugal enhancement of infectivity, reported by Osborn and Walker in 1968, is an
242
HUDSON,
MISRA
1000
g
500
Fraction
No.
FIG. 4. CsCl equilibrium centrifugation of PHlTdRand [3H]UR-labeled nucleic acids from MCV. Nucleic acid was extracted from the virus specimens described in the legend to Table 8, and centrifuged for 72 hr in CsCl (initial density 1.71 g/ ml) in the SW 50.1 rotor at 35,000 rpm at 20”. Fractions were collected from the bottom of each tube onto fiberglass filters. The latter were washed twice with 5% trichloroacetic acid and once with 95% ethanol and their radioactivity was measured. No radioactive pellets could be detected in the tubes. 0, [3HJUR-labeled MCV; labeled 1-16 hr p.i. (Sample B); 0, [3HlUR-labeled MCV; labeled 16-40 hr p.i. (Sample CL The arrow indicates the peak of PHJTdR-labeled MCV nucleic acid (Sample A).
inherent property of murine cytomegalovirus particles, and possibly also of human cytomegalovirus. The evidence may be summarized as follows: (i) MCV obtained from salivary glands, spleen, and mouse embryo cultures shows centrifugal enhancement. (ii) Simultaneous infection of different susceptible cell lines by MCV gives rise to similar c/s ratios. (iii) Other studies (Hudson et al., accompanying manuscript) have shown that both multicapsid and single-capsid virions display the property. (iv) More than 20 different plaque-purified strains of MCV show centrifugal enhancement. Only the “virulent” strain of MCV gave a low c/s ratio (10) of the same order as HSV-1 and HSV-2, but the high c/s ratio was obtained after several in vitro passages in mouse embryo cells. (v) Purified MCV still retains the property. (vi) Over a range of virus dose of four orders of magnitude, there was a constancy of c/s ratio, with linear PFU-
AND
MOSMANN
dose relationships for both modes of infection. (vii) uv-inactivation curves for centrifugal and standard PFU were similar over six orders of magnitude, the c/s ratios never falling below 18 for any samples. (viii) Electron microscopy failed to reveal any particles other than MCV. (ix) No additional DNA- or RNA-containing particles could be detected in MCV preparations after a variety of radioisotope labeling regimens. The small amount of labeled uridine that became associated with purified MCV was ultimately located as an integral part of the viral DNA. Other workers have reported a conversion of uridine into deoxycytidine residues of HSV-DNA (Hirsch and Vonka, 1974; Biswal et al., 1974) and possibly this happens also in MCV-infected cells. Thus in order to express maximum infectivity potential in susceptible cells, MCV requires the aid of a centrifugal field. Discussion of the mechanism responsible for this effect invites much speculation, but it could conceivably involve a membrane-mediated change in the cell’s response to the virus, or a disorganization of certain organelles which normally control the virus. A simple increase in penetration of virions is not the explanation, since centrifugation only causes an approximately threefold increase in penetration into cytoplasm and nuclei, similar to HSV-1. It is possible that most MCV particles entering the cells under standard infection conditions go into a nonreplicating state. If this is so, then it should be possible to influence this state, and hence the c/ s ratio, by manipulating the physiological condition of the cells. This could explain why the c/s ratio varies over a wide range (18 to 85) between different experiments using different cultures. Further studies are being conducted along these lines, in the hope of elucidating the mechanism of centrifugal enhancement, and for the practical reason of finding an alternative procedure to centrifugation for obtaining maximum virus infectivity. Even with the aid of a centrifugal assay, however, normal yields of MCV from mouse embryo cultures are only 50-200 PFU/cell (2-10 PFU per cell without centrifugation). Elec-
CYTOMEGALOVIRUS
tron microscopy of MCV-infected cells suggests that this value represents only a small fraction of total virions assembled. The discrepancy may be due largely to the preponderance of multicapsid virions (Hudson et al., accompanying manuscript), each of which may contain several potentially infectious capsids, but because of their common envelope such a virion can only act as a single infectious entity. Furthermore, the large size of these multiple forms may confer a greater degree of fragility upon them, and if the naked capsids are not infectious then infectivity will be lost. These factors may also explain the relative ease with which infectious MCV can be obtained from salivary glands, since only single-capsid virions were found in homogenates of these glands (Hudson et al., accompanying manuscript). The human cytomegalovirus (HCV), at least the AD169 strain, also gave rise to centrifugally induced infectious centers in human fibroblast cultures, under conditions in which HSV-1 did not. Although we did not pursue the cause of this effect for the HCV, this may indicate ,a general tendency of cytomegaloviruses, which do seem to share certain biological properties (Plummer, 19731, to go into some kind of nonreplicating state within tibroblasts de-
243
INFECTIVITY
rived from their own species. If this is the case, then it is obviously important to investigate in more detail the variety of interactions between cytomegaloviruses and fibroblasts under different conditions. ACKNOWLEDGMENTS The
authors express their thanks to Jessyca zuki and Karen Catherwood for their assistance the project, which was financed by the Medical search Council, Ottawa.
Su in Re-
REFERENCES BISWAL, NICK,
sized 87-99.
N., MURRAY, B. K., M. (1974). Ribonucleotides DNA of herpes simplex
AND
BENYEBH-MEL
in newly synthevirus. Virology 61,
T. R., and HUDSON, J. B. (1973). Some properties of the genome of murine cytomegalovirus (MCV). Virology 54, 135-149. OSBORN, J. E., and WALKER, D. L. (1968). Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2, 853858. OSBORN, J. E., and WALKER, D. L. (1971). Virulence and attenuation of murine cytomegalovirus. Inf. Zmm. 3, 228-236. PLUMMER, G. (1973) Cytomegaloviruses of man and animals. Prog. Med. Virol. 15, 92-125. ROSS, L. J. N., WILDY, P., and CAMERON, K. R. (1971). Formation of small plaques by herpes viruses irradiated with ultraviolet light. Virology 45, 808-812. MOSMANN,
VIROLOGY
(1976)
Cytomegalovirus Infectivity: Analysis of the Phenomenon Centrifugal Enhancement of Infectivity J. B. HUDSON,’ Department
V. MISRA,
of Microbiology,
AND
T. R. MOSMANN
University of British Columbia, British Columbia, Canada Accepted
February
of
Vancouver
V6T
1 W5,
6,1976
The phenomenon of centrifugal enhancement of infectivity was examined in some detial for the murine cytomegalovirus (MCV). Centrifugal enhancement (meaning that application of virus to monolayer cultures with the aid of a centrifugal field resulted in a ZO- to 80-fold increase in infectious centers), was observed in mouse embryo cultures from three different strains of mice, in 3T3 cells, and in rat kidney cells. Three different strains of MCV and 20 plaque-purified strains of MCV all showed the property, as did the one strain of human cytomegalovirus examined. In contrast, herpes simplex virus type 1 and type 2 did not. Centrifugal enhancement could not be explained by increased penetration of MCV or its DNA into cells and their nuclei. Other experiments involving PFU-dose response, uv-inactivation kinetics, electron microscopy, and a variety of labeling regimes with thymidine and uridine isotopes, ruled out the presence of interfering microorganisms. It is concluded, therefore, that the property of centrifugal enhancement is an inherent property of MCV particles, and furthermore, it is suggested that some cytomegaloviruses may have a tendency to enter into a nonreplicating state in homologous fibroblasts.
tures “uncharacteristic” of cytomegaloviruses, such as an unusually large genome (Mosmann and Hudson, 19731, the preponderance of multicapsid virions (Hudson et al., accompanying manuscript), and the reversible attenuation of infectivity following alternate in uivo and in vitro propagation (Osborn and Walker, 1971). Thus, the phenomenon of centrifugal enhancement of infectivity could be just another unusual feature of MCV. We have investigated the phenomenon of centrifugal enhancement in more detail, and extended the study to the human cytomegalovirus. Herpes simplex was used as a control virus not displaying the property. Although the mechanism of the effect is still not understood, the present results indicate that the property (i) is inherent in all MCV particles; (ii) cannot be explained by the presence of an interfering agent, and (iii) is probably also a property of at least one strain of human cytomegalovirus.
INTRODUCTION
Cytomegaloviruses (salivary gland viruses) are considered to be members of the herpes virus genus, although they are often grouped separately from other herpes viruses because of certain characteristic biological features (Plummer, 1973). For example, the cytomegaloviruses generally replicate relatively slowly in cell cultures and yield relatively few infectious particles when assayed by conventional techniques. In order to overcome the latter problem, Osborn and Walker (1968) applied murine cytomegalovirus (MCV) preparations to monolayer cultures with the aid of a centrifugal field, and found an increase of 20- to 80-fold in the number of infectious particles. No such effect was shown by herpes simplex or pseudorabies viruses. Other studies, however, have revealed that MCV possesses certain feaI Author dressed.
to whom
reprint
requests
should
be ad235
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
236
HUDSON, MATERIALS
AND
MISRA
METHODS
Cells Mouse embryo cultures were prepared by standard methods, and used after one to three passages in vitro. Mouse 3T3 cells and H.ep.2 cells were obtained from Flow Laboratories, human WI-38 cells from Microcan Research (Calgary), the rat NRK line from Dr. V. Klement (University of Southern California), and human skin fibroblast cells from Dr. H. Stich (U.B.C. Cancer Research Unit). All cultures were propagated in Dulbecco’s modified MEM supplemented with 20 pg/ml of gentamitin, 0.25 pglml of fungizone, and 5 or 10% fetal calf serum, in a well-humidified atmosphere containing 5% CO, at 37”. Viruses
The murine cytomegalovirus (MCV) strain used in most of the studies was the Smith strain, obtained from the American Type Culture Collection. Fresh stocks of the virus were prepared every 6 to 9 months by low multiplicity passage (co.01 PFU/cell) in Swiss mouse embryo cultures. Large quantities of virus were readily obtained by inoculating stock virus (at less than 0.01 PFU/cell) into roller bottles of mouse embryo cultures. The “virulent” strain of MCV (designated K181, from Dr. June Osborn, University of Wisconsin) was passaged twice in mouse embryo cultures to yield enough virus for examination. The Apodemus sylvaticus (field mouse) strain of MCV, obtained from Dr. Cordon Plummer (Loyola University), was treated in the same way. Herpes simplex virus (HSV) was the type 1, P strain, described previously (Mosmann and Hudson, 1973). The MS strain of HSV-2 was obtained from the American Type Culture Collection. These were passaged in H.ep.2 cells or mouse embryo cultures. The AD169 strain of human cytomegalovirus was obtained from the American Type Culture Collection, and was propagated in WI-38 cells. Viruses were purified by the procedure involving differential centrifugation and nuclease treatment, as
AND
MOSMANN
described 1973).
before (Mosmann
and Hudson,
Plaque purification of MCV. MCV (Smith strain) was purified by repeated centrifugal infection or repeated standard infection and isolation of well-separated plaques. In order to provide sufficient virus for study, the third passage involved low multiplicity infection (less than 0.001 PFU/cell) without an overlay medium, and the cell-free fluids were collected when most of the cells had become infected (3 to 5 days after infection). Plaque assay. MCV was titrated by either the centrifugal or the standard method of inoculation (Osborn and Walker, 1968). One or two milliliters of virus in MEM + 2% serum (for 35- or 50mm diameter plastic dishes, respectively) were added to drain, freshly confluent, mouse embryo monolayers. The dishes were then either centrifuged at 800g for 30 min in the International CS centrifuge, or allowed to stand at ambient temperature for 30 min. The inocula were removed and replaced with either 2 ml per 35-mm dish or 4 ml per 60-mm dish, of overlay, which consisted of MEM plus 0.5% agarose, 5% fetal calf serum, 20 pg/ml of gentamicin, and occasionally 50 units per ml of mycostatin. Plaque counts of the order of 500 to 5,000 were enumerated under the microscope 36-48 hr after infection, and less than 500 per 35-mm dish were counted macroscopically, without staining, after 3 to 6 days. Herpes simplex and human cytomegalovirus were assayed in an analogous fashion, on appropriate cell monolayers. Cell Fractionation
Monolayers of uninfected, MCV-infected, or HSV-infected, cells were washed twice with ice-cold RSB+ (0.1 M NaCl, 0.01 M Tris-chloride, pH 7.4, 0.015 M MgCl&, then with RSB (0.01 M NaCl, 0.01 it4 Trischloride, pH 7.4, 0.0015 M MgCl,). The cells were allowed to swell for 15 min in RSB, scraped off the dish, and homogenized in a Dounce homogenizer to disrupt the cells. This last process was always monitored by phase contrast microscopy to ensure complete disruption of cells with preservation of nuclear integrity. In some
CYTOMEGALOVIRUS
experiments, the washed monolayers were exposed to RSB containing 1% NP40 (Nonidet P40) for 15 min at 4”. The lysates were than shaken from the culture dishes. Nuclei were sedimented at 1000 g for 10 min and washed once. The supernatant from the first centrifugation was used as cytoplasmic extract. Control experiments with both uninfected and MCV-infected cells, containing prelabeled cellular DNA (13Hlthymidine), showed 1% or less of this label in the final cytoplasmic extract obtained by either technique, indicating preservation of nuclei. Phase contrast microscopy revealed little or no obvious contamination of nuclei by cytoplasmic fragments. Ultraviolet
Irradiation
This was samples of distance of germicidal
accomplished by placing l.O-ml virus in 50-mm dishes, at a 20 cm from a Sylvania 15 W lamp.
Nucleic
of Virus
Acid Extraction
Nucleic acid was extracted from virus samples by the pronase-SDS technique described in detail elsewhere (Mosmann and Hudson, 1973). RESULTS
Comparison of Centrifugal Infectivity
and Standard
In the presentation of these results, the term “centrifugal-PFU” refers to plaques obtained following centrifugation of the monolayers in the presence of virus, whereas “standard-PFU” denotes omission of a centrifugal field. Details of the techniques are described in the Materials and Methods section. Initially we repeated many of the experiments reported by Osborn and Walker (1968), using our tissue-culture passaged MCV, and obtained essentially identical results. Over a period of about 3 years, with many different virus preparations, the ratio of centrifugal PFU to standard PFU has varied randomly between extremes of 15 and 85, most values falling within the 25-50 range. This value is referred to as centrifugal enhancement or c/s
231
INFECTIVITY
ratio. Plaque morphology is identical in the two modes of infection comprising characteristic-looking rounded cells retracting from their neighbors and leaving a central clear hole in the monolayer which can be visualized easily without straining. The rate of growth of the plaques is similar, although in older mouse embryo cultures, the initiation of a standard plaque may lag by 24 hr. Virus which did not initially adsorb to the cells following centrifugal or standard inoculation, still displayed centrifugal enhancement upon infection of a second set of cultures. This result argues against the possibility of selective uptake of function” ally different virions, i.e., selective uptake of virions giving either standard or centrifugal modes of infection. Centrifugation of the virus or cultures separately before their mixing did not allow maximum expression of infectivity. The simultaneous presence of both components was required, although centrifugation after standard inoculation did increase plaque numbers. Centrifugal Enhancement Cell Types
in
Different
Most of the experiments described utilized embryo cultures from random bred Swiss mice, the cultures being used after one to three passages in vitro with similar results. Centrifugal enhancement was not unique to these cells, however, since inbred Balb/c and ICR embryo cultures also yielded centrifugal enhancement, as shown in Table 1. Furthermore 3T3 cells and the rat kidney cell line NRK gave the same results (Table 2). It should be pointed out that because the c/s ratio varies randomly from one experiment to another (presumably due to “physiological” variables), strict comparison between virus preparations or cell cultures, can only be made by simultaneous infections. Absolute titers and cls ratios can vary to some extent if a virus preparation is assayed over a period of several days on the same set of cultures. Although 3T3 cells gave a similar virus titer to embryo cultures, they were not used for routine virus assays
238
HUDSON, TABLE
MISRA
1
Other Viruses
MCV INFECTIVITY FROM
IN MOUSE EMBRYO CULTURES DIFFERENT STRAINS OF MOUSE
Embryonic fibroblasts from Swiss (randombred) Balblc ICR
PFU/ml (centrifugal) 2.86 x 106
22.3
2
STRAIN) INFECTIVITY CELL TYPES
Cell line ME 3T3 NRK (normal rat kidney line
42.3 35.8
PFU to standard PFU. TABLE
MCV (SMITH
c/s ration
3.60 x lo6 3.04 x 106
(1Ratio centrifugal
PFU/ml (centrifugal) 2.25 x 108 6.75 x 10’ 3.25 x IO’
IN DIFFERENT
Ratio:c/s 18.5 18.0 26.0
since they had a very narrow range of growth over which plaques were visible. Thus, 3T3 cultures 1 day past confluency no longer yielded discernible plaques. The rat kidney cells gave smaller MCV titers, and the plaques took longer to develop, which may reflect insufficient adaptation to the rat cells. Therefore mouse embryo cells were used routinely for plaque assays. Plaque-Purified
AND MOSMANN
The experiments so far were performed with the Smith strain of MCV. Table 4 shows that the Apodemus isolate of MCV also displayed centrifugal enhancement. In contrast, the K181 strain of MCV initially showed a low c/s ratio, similar to HSV-1 and HSV-2. This is interesting because the K181 strain is more virulent than the Smith strain toward mice (June Osborn, personal communication). However, after four passages in mouse embryo cultures, the c/s ratio of K181 rose to 18.0. Herpes simplex c/s ratios were always less than 8.0, and usually less than 5.0 (Table 51, these values being explicable in terms of a simple increased penetration into the cells under a centrifugal field (see below). Human cytomegalovirus (HCV, strain AD169) also showed centrifugal enhancement when assayed in human fibroblasts. These results are summarized in Table 6. In three out of the four tests centrifugal enhancement was evident, whereas HSV-1 TABLE INFECTIVITY
PFU/ ml (x 10-B
Twenty plaque-purified strains of MCV were derived, 10 by repeated centrifugal infection and 10 by repeated standard infection. Table 3 shows that all 20 strains displayed the property of centrifugal enhancement, the c/s ratios ranging from 18 to 70, with mean values of 48 and 35, respectively, for centrifugaland standardderived strains. There was no obvious difference between the two strain categories. Two strains were assayed again later and, although both showed centrifugal enhancement, the values were different, illustrating the apparent random nature of the c/s ratio. It was therefore concluded that the property of centrifugal enhancement was not attributable to genetic heterogeneity in the virus population.
tio
ard strains
20.3 48.3 18.1 43.5 37.0 28.0 60.0 43.8 47.0 48.8 39.5
Sl s2 s3 s4 s5 S6 s7 S8 s9 SlO mean
TABLE VIRUS
I
OF PLAQUE-PURIFIED MCV ,:is Ra- StandPFU/ cls
1.42 1.50 0.32 1.63 1.35 1.40 0.72 0.70 0.40 0.34
MCV
3
INFECTIVITY
Virus MCV (Smith) MCV (Apodemus) MCV (Osborn-K181) HSV-1 (Strain P) HSV-2 (Strain MS)
4
IN MOUSE
ml (x 10-S)
ratio
0.60 0.18 1.41 3.25 1.49 1.38 2.07 3.08 2.88 2.92
52.1 70.0 46.5 38.4 42.4 39.0 18.8 48.9 38.4 66.4 46.1
EMBRYO
CELLS
Ratio: Centrifugal PFU/standard PFU (c/s) 18.0 23.6 5.2 3.7 5.6
CYTOMEGALOVIRUS TABLE HSV Cell
line
ME
PFU/ml (centrifugal)
HSV-1 HSV-2 HSV-1 HSV-2
NRK
3.8 2.0 2.0 2.2
TABLE INFECTIVITY
OF HCV
Test 1 2 3 4 5
Cell
TABLE
5
INFECTIVITY
Virus
type
WI-38 WI-38 Human skin iibroblasts Human skin fibroblasts Human skin flbroblasts
x x x x
lo5 104 10” 10”
Ratio: C/S
4.2 3.9 2.9 5.6
7
ENHANCEMENT OF [3H]TdR-LanELEn VIRUS UP BY THE NUCLEUS BY CENTRIFUGAL INO~ULATIO@ Fraction
Centrifugal ‘T;;’
IN HUMAN
CELLS
Virus
c/s ratio
HCV HCV HCV
25.0 25.0 7.5
HCV
30.0
HSV-1
3.7
of MCV into Cells and Nuclei
One obvious explanation for centrifugal enhancement would be a greatly increased uptake of virus into the cells, or into the nuclei. This was initially considered plausible in view of the predominant multiple capsid virions in MCV populations (Hudson et al., accompanying manuscript). In order to test this hypothesis, MCV was labeled with tritiated thymidine. Herpes simplex type 1 was similarly labeled and used for comparison. The results of one experiment are displayed in Table 7, from which it can be seen that penetration into cells and into their nuclei was increased two- to threefold by centrifugation, for both viruses. The same result was obtained for 3T3 cells, using both of the methods of cell fractionation described in the Materials and Methods section. Thus the infectivity of MCV is increased by an additional order of magnitude by applying
TAKEN
Enhancement
Nuclear fraction
1946 2636
) 2.6-fold i 3.2-fold
Cytoplasmic fraction
1810 4752
I 1 2.8-fold i 2.5-fold
6
AND HSV
in these cells gave a ratio of only 3.7. The HCV plaques developed slowly, being invisible to the naked eye until about 9 days after infection, but the c/s ratio remained constant from their first appearance under the microscope (4-5 days p.i.) until degeneration of the monolayers set in after 3 weeks. Penetration
239
INFECTIVITY
o Monolayers of mouse embryo cells infected with l”H]TdR-labeled MCV or HSV by either the standard or centrifugal method were washed twice with ice-cold RSB+, then with RSB. The cells were allowed to swell for 15 min at 4” in RSB, containing 0.1% NP40, scraped off the dish, and homogenized in a Dounce homogenizer to disrupt the cells. Nuclei were sedimented at 1000 g for 10 min, and washed once with RSB. The supernatant from the first centrifugation was used as cytoplasmic fraction.
a centrifugal field, over the amount accounted for in terms of increased physical uptake of virus particles. The next series of experiments was designed to investigate the possibility of an interfering agent, virus or some other particle, which might normally suppress MCV replication except under centrifugal conditions. Proportionality and Virus
of Plaque-Forming Dose
Units
MCV produces very small though distinct plaques, and as a consequence, it is possible to count between 1.0 and 5000 plaques on a 35mm dish, up to 500 with the naked eye, and more than this under a microscope. This feature enabled us to examine c/s ratios over four orders of magnitude. Figure 1 shows the results for one such test, from which it is evident that the ratio, in this case 22.5, was essentially constant over this range, the dose response being linear for both centrifugal and standard infections. Ultraviolet
Irradiation
of MCV
Other enveloped animal viruses often displayed complex kinetics of uv-inactivation (e.g., Ross et al., 1971). Likewise MCV
240
HUDSON,
MISRA
AND
MOSMANN
by incubating infected cells at various times with 13Hluridine, and purifying MCV from the cell free fluids by the norwhich involved both mal procedure, DNAse and RNAse treatment. The results, in Table 8, show that relatively little
io4-
103it 5 z f B L IOZ-
IO-
I 10-7
I 10-b Virus Concentration
FIG. 1. Plaque stock of MCV was duplicate by both niques, on 50-mm
I 10-s (Relative
I lo-4 ]
number versus MCV dilution. A serially diluted and assayed in centrifugal and standard techdishes of mouse embryo cells.
does, as illustrated in Fig. 2. Both centrifugal and standard PFU decayed at similar rates, such that the c/s ratio at all uv doses was greater than 20, although the actual value varied randomly among the samples tested. We do not know if multipleand single-capsid virions have the same uv sensitivity; nevertheless, the range of uv doses employed was enough to kill all infectious entities, so that if any interfering particles were present, then they must have had the same uv sensitivity as MCV. Attempts to Label an Interfering Agent Virions labeled with [3H]- or [14C]thymidine, or 32P, and purified by differential centrifugation and DNAse treatment, always revealed a single species of DNA in sedimentation velocity or equilibrium density gradients (Mosmann and Hudson, 1973). This argues against the possibility of an interfering DNA-containing agent. However, virus purified by our normal procedure still retains the property of centrifugal enhancement. An RNA containing agent was sought
t 1.0
0.5
I I.5
I I 2.0 2.5 UV Dose Imins.)
I 3.0
I 3.5
t 4.0
FIG. 2. Effect of uv irradiation upon MCV infectivity. A stock of MCV (in MEM) was exposed to uv irradiation, and samples were withdrawn at intervals for plaque assay by centrifugal and standard techniques. The B-min samples contained zero PFU. 0, Centrifugal PFU; 0, standard PFU. TABLE INFECTED
Sample
8
OF [$H]TdR
INCOIWRATION
MOUSE
Labeling time (hr p.i.1
EMBRYO
Harvest t$e p.ir)
A. B. C. C.
TdR UR UR UR
label label label label
16-40 1-16 1-16 16-40
13H]UR
AND
40 16 40 40
BY MCV-
CELLS”
cpm per culture Cell-free supernatant
Ensymetreated virus pellet
109,570 7,800 39,650 1 55,250
81,650 <20 14,220 17,500
0 A series of mouse embryo cultures (in 50-mm dishes) were infected with MCV, and at the appropriate times, 5 @Z!i of 13HJTdR or 13HlUR were added per dish. At the indicated times, groups of four dishes were harvested for virus purification.
CYTOMEGALOVIRUS
uridine label and no enzyme-resistant label could be detected in cell free fluids harvested 16-hr postinfection (i.e., before progeny MCV is excreted), but significant amounts were found after this time, when thymidine-labeled MCV is also detected. The labeled viral pellets were examined further by sedimentation velocity in sucrose gradients. Figure 3 shows that most of the labeled material sedimented into the 60% sucrose cushion for samples A, B, and C, although some low molecular weight thymidine label was also evident. There was no significant amount of slower sedimenting particulate material, and in other experiments with thymidineand uridinelabeled MCV preparations, there were no particles that sedimented faster than MCV. Similar preparations were also centrifuged in 20-60% equilibrium sucrose gra-
Fraction
No.
3. Sedimentation velocity of [3H]TdRand 13HlURlabeled MCV. The virus specimens, described in the legend to Table 8, were centrifuged in 5-208 sucrose gradients (w/v, in PBS, 4.5 ml per tube) with a cushion of 0.5 ml of 60% sucrose. Centrifugation was carried out for 30 min at 15,000 rpm in the SW 50.1 rotor at 4”. Fractions were collected directly onto fiberglass filters for radioactive measurement. None of the “pellets” in the bottoms of the tubes contained detectable radioactivity. 0, 13HlTdR-labeled MCV; labeled 16-40 hr p.i. (Sample A); n , [3HlUR-labeled MCV; labeled 1-16 hr p.i. (Sample B); 0, [3H]UR-labeled MCV; labeled 16-40 hr p.i. (Sample C). FIG.
241
INFECTIVITY
dients. MCV showed a broad range of buoyant densities in such gradients, in contrast to the narrow band found for 13H]UR-labeled murine sarcoma virus (Kirsten strain, derived from transformed NRK cells). This heterogeneity in MCV density presumably reflects the presence of multiple-capsid virions (Hudson et al., accompanying manuscript). Nevertheless, uridine- and thymidine-labeled MCV profiles were essentially coincident, implying that the two isotopes were incorporated into the same particles. In order to investigate the nature of the uridine label in MCV, nucleic acids were extracted from the same preparations used for Fig. 3, and centrifuged to equilibrium in CsCl gradients. Figure 4 shows that uridine and thymidine labels are coincident, and band at the density expected for MCV DNA (Mosmann and Hudson, 1973). Thus the uridine label is an integral part of the MCV DNA, which could indicate either the presence of RNA bound to or covalently linked to the DNA, or that the uridine had been converted to a deoxyribonucleotide prior to incorporation into progeny DNA. The latter alternative is supported by the observation that the uridine label found in MCV DNA is resistant to pancreatic RNAse and alkaline hydrolysis both in the native and denatured DNA (unpublished results). Although more evidence is required to clarify this point, in the context of the present study it seems justifiable to conclude that our MCV prep arations do not contain significant numbers of any other RNA- or DNA-containing particles. This conclusion is reinforced by our inability to find particles other than typical herpeslike particles in thin sections of MCV-infected cells viewed in the electron microscope. We therefore conclude from these studies that centrifugal enhancement of infectivity is an inherent property of MCV particles. DISCUSSION
The results described in this communication clearly indicate that the property of centrifugal enhancement of infectivity, reported by Osborn and Walker in 1968, is an
242
HUDSON,
MISRA
1000
g
500
Fraction
No.
FIG. 4. CsCl equilibrium centrifugation of PHlTdRand [3H]UR-labeled nucleic acids from MCV. Nucleic acid was extracted from the virus specimens described in the legend to Table 8, and centrifuged for 72 hr in CsCl (initial density 1.71 g/ ml) in the SW 50.1 rotor at 35,000 rpm at 20”. Fractions were collected from the bottom of each tube onto fiberglass filters. The latter were washed twice with 5% trichloroacetic acid and once with 95% ethanol and their radioactivity was measured. No radioactive pellets could be detected in the tubes. 0, [3HJUR-labeled MCV; labeled 1-16 hr p.i. (Sample B); 0, [3HlUR-labeled MCV; labeled 16-40 hr p.i. (Sample CL The arrow indicates the peak of PHJTdR-labeled MCV nucleic acid (Sample A).
inherent property of murine cytomegalovirus particles, and possibly also of human cytomegalovirus. The evidence may be summarized as follows: (i) MCV obtained from salivary glands, spleen, and mouse embryo cultures shows centrifugal enhancement. (ii) Simultaneous infection of different susceptible cell lines by MCV gives rise to similar c/s ratios. (iii) Other studies (Hudson et al., accompanying manuscript) have shown that both multicapsid and single-capsid virions display the property. (iv) More than 20 different plaque-purified strains of MCV show centrifugal enhancement. Only the “virulent” strain of MCV gave a low c/s ratio (10) of the same order as HSV-1 and HSV-2, but the high c/s ratio was obtained after several in vitro passages in mouse embryo cells. (v) Purified MCV still retains the property. (vi) Over a range of virus dose of four orders of magnitude, there was a constancy of c/s ratio, with linear PFU-
AND
MOSMANN
dose relationships for both modes of infection. (vii) uv-inactivation curves for centrifugal and standard PFU were similar over six orders of magnitude, the c/s ratios never falling below 18 for any samples. (viii) Electron microscopy failed to reveal any particles other than MCV. (ix) No additional DNA- or RNA-containing particles could be detected in MCV preparations after a variety of radioisotope labeling regimens. The small amount of labeled uridine that became associated with purified MCV was ultimately located as an integral part of the viral DNA. Other workers have reported a conversion of uridine into deoxycytidine residues of HSV-DNA (Hirsch and Vonka, 1974; Biswal et al., 1974) and possibly this happens also in MCV-infected cells. Thus in order to express maximum infectivity potential in susceptible cells, MCV requires the aid of a centrifugal field. Discussion of the mechanism responsible for this effect invites much speculation, but it could conceivably involve a membrane-mediated change in the cell’s response to the virus, or a disorganization of certain organelles which normally control the virus. A simple increase in penetration of virions is not the explanation, since centrifugation only causes an approximately threefold increase in penetration into cytoplasm and nuclei, similar to HSV-1. It is possible that most MCV particles entering the cells under standard infection conditions go into a nonreplicating state. If this is so, then it should be possible to influence this state, and hence the c/ s ratio, by manipulating the physiological condition of the cells. This could explain why the c/s ratio varies over a wide range (18 to 85) between different experiments using different cultures. Further studies are being conducted along these lines, in the hope of elucidating the mechanism of centrifugal enhancement, and for the practical reason of finding an alternative procedure to centrifugation for obtaining maximum virus infectivity. Even with the aid of a centrifugal assay, however, normal yields of MCV from mouse embryo cultures are only 50-200 PFU/cell (2-10 PFU per cell without centrifugation). Elec-
CYTOMEGALOVIRUS
tron microscopy of MCV-infected cells suggests that this value represents only a small fraction of total virions assembled. The discrepancy may be due largely to the preponderance of multicapsid virions (Hudson et al., accompanying manuscript), each of which may contain several potentially infectious capsids, but because of their common envelope such a virion can only act as a single infectious entity. Furthermore, the large size of these multiple forms may confer a greater degree of fragility upon them, and if the naked capsids are not infectious then infectivity will be lost. These factors may also explain the relative ease with which infectious MCV can be obtained from salivary glands, since only single-capsid virions were found in homogenates of these glands (Hudson et al., accompanying manuscript). The human cytomegalovirus (HCV), at least the AD169 strain, also gave rise to centrifugally induced infectious centers in human fibroblast cultures, under conditions in which HSV-1 did not. Although we did not pursue the cause of this effect for the HCV, this may indicate ,a general tendency of cytomegaloviruses, which do seem to share certain biological properties (Plummer, 19731, to go into some kind of nonreplicating state within tibroblasts de-
243
INFECTIVITY
rived from their own species. If this is the case, then it is obviously important to investigate in more detail the variety of interactions between cytomegaloviruses and fibroblasts under different conditions. ACKNOWLEDGMENTS The
authors express their thanks to Jessyca zuki and Karen Catherwood for their assistance the project, which was financed by the Medical search Council, Ottawa.
Su in Re-
REFERENCES BISWAL, NICK,
sized 87-99.
N., MURRAY, B. K., M. (1974). Ribonucleotides DNA of herpes simplex
AND
BENYEBH-MEL
in newly synthevirus. Virology 61,
T. R., and HUDSON, J. B. (1973). Some properties of the genome of murine cytomegalovirus (MCV). Virology 54, 135-149. OSBORN, J. E., and WALKER, D. L. (1968). Enhancement of infectivity of murine cytomegalovirus in vitro by centrifugal inoculation. J. Virol. 2, 853858. OSBORN, J. E., and WALKER, D. L. (1971). Virulence and attenuation of murine cytomegalovirus. Inf. Zmm. 3, 228-236. PLUMMER, G. (1973) Cytomegaloviruses of man and animals. Prog. Med. Virol. 15, 92-125. ROSS, L. J. N., WILDY, P., and CAMERON, K. R. (1971). Formation of small plaques by herpes viruses irradiated with ultraviolet light. Virology 45, 808-812. MOSMANN,