Quantitation of the interaction between adenovirus types 2 and 5 and microtubules inside infected cells

105, 265-269 (1980)

VIROLOGY

Quantitation

BARBARA

of the Interaction Microtubules

D. MILES,**’ ROBERT

between Adenovirus Types 2 and 5 and inside Infected Cells

RONALD B. LUFTIG,S JAMES A. WEATHERBEE,” R. WEIHING,” AND JOSEPH WEBERt

*Worcester Foundationfor Experimental Biology, Shmwsburg, Massachusetts 01545, tlhivemity of Slwrbruoke, Sherbrooke, Quebec, Canada, JlH G& and *Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina 29208 Accepted May 16, 1980 Electron microscopy of thin sections of HEp-2 cells at 1 h after infection with adenovirus type 5 showed that 31% of the virus particles observed were associated with cytoplasmic microtubules. A similar examination of cells infected with a temperature-sensitive mutant (tsof adenovirus type 2, using particles produced at the permissive temperature 1:33”), showed a similar fraction (25-341) of the viral particles associated with microtubules at 1 or 6 hr after infection. In contrast, when HEp-2 cells were infected with particles produced from cells infected with ts-1 at the restrictive temperature (ts-1:39”), most of the particles (74-9496) observed at 1 to 6 hr postinfection were found enclosed in large vacuoles which were biochemicallv demonstrated to be lysosomes. Only a few particles (2-6%) were associated with m&tubules.

Association of adenovirus with microtubules has been observed in vivo (1) and in vitro (2,3). Electron micrographs of thin sections of HeLa cells examined between 15 min and 2 hr after infection with adenovirus type 5 have shown virus particles close to cytoplasmic microtubules (1). Furthermore, depolymerization of microtubules with vinblastine or colchicine (1, 4) delayed or decreased virus production. Based on these experiments it has been suggested (1) that intracellular adenovirus first interacts specifically with microtubules, and is then transported vectorially to the nucleus. The specific association of adenovirus type 5 with rat brain microtubules first observed by Luftig and Weihing (2) and then Weatherbee et al. (3) using an in vitro binding assay support this model. We now report a quantitative morphological analysis of the association of adenovirus with cytoplasmic microtubules visualized by electron microscopy of thin sections of infected cells. We incorporated a microtubule stabilizing buffer into the fixa1 Author addressed.

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tive, which enhances microtubule visualization threefold (5 ), and quantitated the fraction of virus particles association with microtubules and other cell fractions. We found that adenovirus type 5 and adenovirus type 2 ts-1:33” (6) but not adenovirus type 2 ts1:39” were associated with microtubules more frequently in vivo than would be expected if they were distributed at random in the cytoplasm. HEp-2 cells were propagated in T-75 flasks in Dulbecco’s mod&d Eagle’s medium (DME, GIBCO) plus penicillin (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum (Microbiological Associates). Adenovirus stocks were grown and purified by the procedure of Weber (6). To produce 35S-labeled adenovirus type 2 ts-1:39”, monolayers of HEp-2 cells were infected with ts-1:33” at a multiplicity of approximately 1000 particles per cell. After adsorption of the virus for 90 min at 39”, 25 ml of DME without methionine, containing 5% heat-inactivated fetal calf serum, 100 units/ml each of penicillin and streptomycin, 0.4 mM arginine, 0.02 mlK cold methionine, and 0.25 mCi/dish 35S-methionine was added per T-75 flask. At 30 hr postinfection the 0042-6822/80/11026505$02.00/0 Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form resewed.

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FIG. 1. Examples of adenovirus-microtubule associations in HEp-2 cells 1 hr postinfection’asseen in thin sections of pelleted cells. Adenovirus type 5 is shown in a and b, and adenovirus type 2ts-1:33” in c. Arrows point to areas of virus-microtubule association. Large virus-filled vacuoles are frequently seen in the cytoplasm of HEp2 cells 1 hr after infection with adenovirus type 2 ts-1:&P (d). Magnification: (a) x 105,000; (b) x 183,000; (c) x 111,000; and (d) ~81,000. Bar is 0.1 pm.

medium was aspirated and replaced with fresh medium of the same composition. Virus was purified after 48 hr (6 >. In the experiments reported here, virus

infections were carried out at multiplicities as above. For maximal loading of the virus onto cells, monolayers were prechilled and the virus adsorbed at 4” for 2 hr. Twenty-

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over the 1% value that we can calculate below for the random localization of adenovirus particles within the cytoplasm of a cell. This calculation was made by empirically estimating that microtubules occupy about 2% of the total cytoplasmic area of an average cell profile, using data taken from thin section electron micrographs used in Luftig et al. (5). HEp-2, another human cell line which appears to show a similar distribution of cytoplasmic microtubules in thin sections (B. Miles, unpublished data), was used in these studies because it consistently produced a greater yield of adenovirus than did HeLa cells. From Table 1 it can be seen that about 50% (31 + 18%) of the total adenovirus type 5 particles counted were associated with microtubules or free in the cytoplasm. If this fraction of the virus was randomly associated with the 2% of the cytoplasmic area counted for the microtubules, then we would expect only 1% (2% of 50%) of the virus to be associated with microtubules. Instead, we found 31% of the virus associated with microtubules, suggesting a high specificity of interaction. We further note that this 31% value is a conservative figure because: (a) it neglects binding above and below the plane of the section, (b) the preservation of microtubules although improved may still not be complete, (c) virus in vacuoles may be a noninfectious

five milliliters per T-75 flask of warm DME with 10% fetal bovine serum and 0.4 mM arginine was added, and cells were immediately placed in the humidified, 5% CO*, 37” incubator. After the appropriate incubation time, cells were removed from the flasks with 0.25% trypsin (GIBCO) followed by a neutralizing amount of lima bean trypsin inhibitor (Sigma). Cells were pelleted at 1500 g for 10 min, fixed in 2.5% glutaraldehyde, dehydrated through increasing concentrations of ethanol, and embedded in Epon 812. Thin sections were cut on a Sorvall Porter-Blum MT-2B ultramicrotome, stained with 2% aqueous uranyl acetate and Reynolds lead citrate (11), and examined on a Phillips EM301 electron microscope. HEp-2 cells were infected with WT adenovirus type 5 and sampled 1 hr after infection at 37”. We examined 50 cell sections, counted 232 virus particles, and placed them into one of five categories depending on their cellular location: (a) associated with or in close proximity to microtubules, (b) enclosed in vacuoles, (c) free in the cytoplasmic matrix, (d) outside of the plasma membrane, or (e) associated with the nuclear envelope. As can be seen in Figs. la, b, and c, one or two virus particles are often aligned along a microtubule. We found that 31% of all virus particles seen were associated with microtubules (Table l), a significant increase TABLE

1

LOCATION OF ADENOVIRUS TYPE 5 AND TYPE 2 ts-1 PARTICLES WITHIN HEp-2 CELLS AT 1 OR 6 hr AFTER INFECTION” Microtubules* (%) Ad 5

Vacuoles” (%)

Cytoplasmic matrix (%)

Nuclear envelope (%)

Outside (%)

Total number d

1 hr

31

24

18

6

21

232

Ad 2 (ts-1:33”) 1 hr Ad 2 (ts-1:33”) 6 hr

34 25

13 20

32 30

14 17

7 8

2097 317

6 2

74 94

10 4

0 0

10 0

1895 547

Ad 2 (k-1:39”) Ad 2 (k-1:39”)

1 hr 6 hr

0 Each data point is presented as a percentage mental row. h Microtubule-associated particles were counted r ts-1:39” particles located within lysosome-like d This is the total number of particles counted total number of particles.

of the total number of particles

counted for each experi-

as seen in Figs. la, b, and c. vacuoles as seen in Fig. Id. for each experimental row. The percentages

are per this

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dead-end pathway, (d) the 6% of virus which have reached the nucleus may have been - I8 associated with microtubules previously 1.6 - 16 while being transported, and (e) the 21% of virus still outside the plasma membrane - 14 may be noninfectious, or may subsequently -12% become associated with microtubules after ‘; penetrating the plasma membrane. The ‘O E results shown in Table 1 are also in overall -8” agreement with the morphological distribu0 tion of adenovirus type 5 in HeLa cells -6 reported by Chardonnet and Dales (IS, 1.4>. In the second series of experiments, ts-1, a temperature-sensitive mutant of adenovirus type 2, was used (6, 7). At 39” (the restrictive temperature) ts-1:33”-infected cells produce a normal yield of noninfectious FRACTION NUMBER TOP BOTTOM particles designated ts-1:39”. When these particles are used to infect cells between FIG. 2. Comparison of acid phosphatase activity (0) and “S-labeled virus (0) in fractions from sucrose 33 and 39”, they adsorb to and penetrate the plasma membrane, but fail to uncoat density gradients on which the cytoplasmic organelle at the nucleus, as evidenced by their failure fractions of adeno 2 ts-1:39” infected cells were run. to become slow sedimenting structures as Four confluent T-75 flasks of HEp-2 cells were infected do wild-type virus (10). as previously described with 35S-labeled adenovirus type 2 m-139”; a total of approximately 1.5 million We compared the intracellular distribucounts were used per experiment. Cells were incubated tion of adenovirus type 2 ts-1:33” and ts-1:39” for 1 hr at 37”, scraped from the flask with a rubber virus in the hope that we would discover policeman, pelleted at 1OOOgfor 5 min, and resusa specific defect of intracellular association pended in 10 ml of 8% sucrose with 10M3M disodium of the ts-1:39” virus with the microtubules, EDTA pH 7.0. The following lysosome isolation is a Instead, we found that these h-1:39“ viruses, modification of a technique provided by Samuel Silverwhich do express the mutation, are primarily stein, Rockefeller University (personnel communicataken up into lysosomal vesicles (Table 1). tion). The cells were ruptured in a Dounce homogenizer In contrast, the ts-1:33” virus, which do not for 15 strokes, then centrifuged at 1000 g for 10 min. The supernatant was decanted and cytoplasmic or- express the mutation, were found associated ganelles were pelleted from it at 15,OOOgfor 40 min. with cytoplasmic microtubules to the same This pellet was resuspended in 1.0 ml of 8% sucrose degree as adenovirus type 5 (Table 1). These with 1O-3M EDTA pH 7.0, layered onto a 24-53% (in differences in localization resemble dif0.1 M Tris pH 7.2) continuous sucrose gradient with ferences reported earlier by Chardonnet a 0.5 ml 70% sucrose cushion, and centrifuged at and Dales (14 ) in which adenovirus type 5 40,000 rpm for 1 hr in a Spinco 50.1 rotor at 4”. Twenty was found primarily in cytoplasm in HeLa drop fractions were collected from each gradient. cells, while adenovirus type 7 was found in From each fraction, 0.2 ml was removed and counted phagosomes and lysosomes. In that study, in 10 ml Aquasol (New England Nuclear) in a Beckman LS-330 liquid scintillation counter. The remainder of virus particles found in the cytoplasm were each fraction was frozen and thawed 10x in an ace- not designated as to whether or not they tone and dry ice bath, incubated with 0.5 ml of 0.4 M were associated with microtubules, and P-glycerophosphate (Sigma) in 0.05 M citrate buffer vacuoles were categorized as being either pH. 5.0 for 2 hr at 37”, then precipitated with 2 ml phagocytic of lysosomal in nature. In our of 10% TCA. After 5 min, each sample was cen- EM study we preferred not to make this trifuged at 5000 g for 5 min. Acid phosphatase acdistinction without cytochemical proof, betivity was determined calorimetrically (12 ). To 1 ml of cause of the known heterogeneity in the supernatant, 0.5 ml of Fiske-Subbarow’s molybdate II reagent (3 N sulfuric acid with 2.5% ammonium morphology of lysosomes (15). In the Chardonnet study, 1 hr after infection with molybdate) and 0.2 ml of Fiske-Subbarow reducer (Sigma) was added. After 15 min, the O.D. at 660 adenovirus type 5, 52.8% of the infecting virus was found in the cytoplasmic matrix nm was measured on a Gilford spectrophotometer.

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vs our 66% (microtubule associated + cytoplasmic matrix). They found 34.4% in vacuoles (phagocytic + lysosomal) as compared to our 13%. One hour after infection with adenovirus type 7, Chardonnet found 10.6% in the cytoplasmic matrix and 96.5% in vacuoles vs our 16 and 74%, respectively, for ts1:39”. To determine biochemically whether these virus-filled vacuoles were actually lysosomes, the lysosome fraction was isolated from HEp-2 cells 1 hr after infection with 35S-labeled adenovirus type 2 ts-1:29 by sucrose density gradient centrifugation. Figure 2 summarizes data from two experiments. The lysosome fraction, located by the peak in acid phosphatase activity, coincides with the 35Sor virus peak. Our observations on the intracellular distribution of the ts-1:39” virus have one further similarity to those for adenovirus type 7 infected cells (16). In both cases, particles persist in lysosome-like vacuoles for 6 hr or more, suggesting that these particles are in a dead-end pathway, perhaps because they cannot be transferred from the plasma membrane to the cytoplasm. Our morphological observation that no particles of ts-1:39” were ever seen at the nuclear membrane is also in accord with the biochemical observations of Mirza and Weber (10 >,who found that many viral cores never became associated with or penetrate the nucleus. In fact, our observations suggest that ts-1:39” may follow the “alternative” pathway proposed by Mirza and Weber (IO) in which the virus particles are uncoated in the cytoplasm and only the core structure becomes associated with the nucleus. Further analysis suggests that those few ts-1:39” particles which are available for interaction with microtubules actually do so to about the same degree as do the adenovirus type 5 (31% after 1 hr). This suggestion is based on calculation of the following ratios using data from Table 1 of the ts l-39” particles available for interaction with microtubules (the fraction found associated with microtubules plus the fraction found in the cytoplasmic matrix), 37.5% or [6% t (6 + lo%)] are associated with microtubules at 1 hr after infection and33.3% or [2% t (2 + 4%)1 at 6 hr after infection. Because these calculations are ultimately

based on very small numbers of particles, additional experiments will be necessary to determine the validity of this suggestion. Finally, we recognize a difficulty common to all electron microscope studies of virusinfected cells where the particle-to-infectivity ratio is quite high, namely, that we are only following the fate of the majority of particles and not necessarily the infectious entities. Such studies are important, however, since they establish a working hypothesis for the fate of virus particles inside infected cells. ACKNOWLEDGMENTS We acknowledge support from NIH Grants CA18979 (to RBL), P30-12708 (to M. B. Hoagland), a cancer research scholar award from the American Cancer Society (Massachusetts Division) (to R.R. W.). and the Mimi Aaron Greenberg Cancer Research Institute. This work is in partial fulfillment of the requirements for a Ph.D. at Clark University by one of us (B.D.M.). REFERENCES 1. DALES, S., and CHARDONNET, Y., Virology

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