Kinetics of TMV-RNA synthesis and its correlation with virus accumulation and crystalline viral inclusion formation in tobacco tissue culture

VIROLOGY

41,

?%?--%‘j

Kinetics

(1972)

of T&W-RNA

Accumulation

Synthesis

and

Crystalline

in Tobacco L. E. PELCHER,

and

its Correlation

Viral

Tissue

Inclusion

with Virus Formation

Culture

H. H. MURAKISHI,

AND

Accepted November

15, 1971

J. X. HARTMANN

Tobacco mosaic virus ribonucleie acid (T~~V-RNA) synt,hesis and the formation of complete virus were studied at 1%hr intervals after inoculation of tobacco (Nicotima tabacum L. var. Havana 38) tissue culture. The rate of TMV-RNA synthesis, as measured by the incorporation of uridine-3H into viral nucleic acid, increased in a nearly linear manner during the first 69 hr after inoculation, reaching a peak during the 48-6@hr period. At this time the rate of viral RNA synthesis was approximately 6 times that of the 12-24-hr period. After the first 60 hr, the rate of viral RNA synthesis declined. The peak rate of accumulation of complete virus occurred during the 4%60-hr period, the period of most rapid viral RNA synthesis. Up t,o 48 hr after ino&&ion, crystalline viral incl~ions were rarely observed, but at 72 hr large aggregates of crystal-bearing cells were present. After this time no incresse in the number of crystal-bearing aggregates was observed. It is likely that plaamodesmata allow for rapid spread of the virus throughout inoculated cell aggregates. The close correlation between viral RNA synthesis, accumulation of complete virus and crystal formation in inoculated tissue culture indicate that the maximum rate of virus synthesis occurred during the first 60 hr. After this time virus synthesis declined and further spread of virus was probably limited to division of virus-infected cells.

(Kassanis, 1967). The development of a plant virus-plant tissue culture system with an improved efficiency of inoculation and a higher virus titer offers a valuable tool for study of bioche~cal events associat& with virus infection (Pelcher and Murakishi, 1971; Beachy and Murakishi, 1971). The adaptation of the polyacryla~de gel technique for the separation of nucleic acids of plant origin (Loening and Ingle, 1967) and its recent application to the study of nucleic acid synthesis in ~rus-iIlf~t~ plants (Fraser, 1969; Hirai and Wildman, 19G9) suggested that this technique would be useful in st,udying the kinetics of viral nucleic acid synthesis in vir~-inoculate plant tissue culture. Zech (19.52) and Nilsson-Tillgren et al.

In recent publications we reported on the development of a plant tissue culture system useful for the study of plant virus replication and the effects of virus infection on the host cells (Murakishi et al., 1970, 1971; Beachy and Murakishi, 1971). Though viral nucleic acid synthesis (Diener, 1962; Kubo, 1966; Hirai and Wildman, 1967; Nilsson-Tillgren, 1969), and its effects on host cell nucleic acid synthesis (Babos, 1969; Fraser, 1969; Hirai and Wildman, 1969) have been studied extensively in the intact plant, little is known of such events following inoculation of a plant tissue culture with a plant virus. The major drawbacks to such studies with plant tissue culture have been the low efficiency of infection and the low virus titer attained 787

788

PELCHER,

MURAKISHX,

(1969) have studied the formation of crystalline viral inclnsions in tobacco leaves following inoculation with TMV. These workers suggested that the apparent synchronous formation of viral inclusions in certain areas of the leaf indicated that all the cells in these areas were in the same stage of i~lfection. Obse~atio~ by ~urakishi et ~2. (1970) indicate that a high percentage of tissue culture cells inoculated with TMV contain crystalline viral inclusions after 7 days. In the study reported here an attempt was made to follow the kinetics of viral RNA synthesis in infected cells and to correlate this with the accumulation of complete virus and the formation of crystalline viral inclusions. ~AT~~~ALS

Inoculation

AND METE~ODS

and uridine-3H

feeling

of tis-

sue culture cells. A pigmented cell culture derived from stem tissue of tobacco (Nicotiana tabacum L. var. Havana 38) was used throughout this study. The cells were maintained and inoculated as previously described (Murakishi et al., 1971). A common strain of TMV was used for inoculation at a concentration of 150 pg/ml of cell suspension. Cells were inoculated in l-g batches suspended in 3 ml of liquid medium. Cells were then pooled and washed with 30 ml of fresh medium. Three-gram aliquots of cells were transferred to a 4 cm disk of Whatman No. 4 filter paper and transferred to a petri plate containing 20 ml of medium solidified with 1% agar. The cells were incubated at approximately 25” under light provided by Gro-lux fluorescent lamps. At 12-hr intervals after inoculation the cells were transferred on the filter paper to a 5-cm petri plate containing 2 ml of fresh liquid medium supplied with 50 PCi of uridine-3H general label (420 mCi/mM) and incubated under light for 12 hr. During the labeling period the medium was constantly agitated. Control cells shaminoculated with phosphate buffer (0.1 M, pH 7.6) were treated in like manner. Inoculated and control cells were transferred from

AND HARTMANN

the agar medium to uridine-3 containing liquid medium every I2 hr throughout the 168-hr period studied. After each labeling period the cells were washed with 20 ml of cold 0.25 M sucrose and divided into two 1.5-g (wet weight) aliquots, one of which was frozen at - 35’ to be used for extraction of complete virus; the other aliquot was immediately extracted with phenol to isolate cellular and viral nucleic acids. Extraction of total cellular and viral nucleic actids. Total nucleic acids were extracted from 1.5 g of cells by grinding (20 strokes) in a TenBroeck glass homogen~er containing 10 ml of distilled water, 0.5 % sodium dodecyl sulfate (SDS) w/v, and 0.1% disodium naphthlene sulfonate (w/v). Immediately after homogenization, 10 ml of watersaturated phenol containing 10 % m-cresol (v/v) and 0.1% 8-hydroxyquinoline {w/v) was added (Kirby, 1965). The homogenate was then mixed for 10 min on a Model K-506 J Vortex mixer (Scientific Industries, New York). The phases were separated by cen~,rifugation at 18,500 g for 10 min. All steps in the extraction procedure were carried out at 4”. The aqueous phase was removed, made 0.3 M with respect to NaCl, and again extracted with the phenol mixture for 5 min. This procedure was repeated t,wicc. Nucleic acids were precipitate from the aqueo~ls phase by addition of 2.5 volumes 95 % ethanol, stored at -35” for 12 hr, and collected by centrifugation at 20,000 g for 20 min. The pellet was washed once with 75 % ethanol ~ont~ning 0.1% NaCl (w/v) and once with 3.0 M sodium acetate p.II 6.0. The fmal pellet was then dissolved in clectrophoresis buffer (0.04 M Tris, 0.02 M sodium acetate, 0.001 M sodium EDTA brought to pH 7.8 with glacial acetic acid) containing 5 % sucrose (w/v). The ultraviolet absorption spectra were then determined in a Beckmann DB spectrophotometer. The relative optical densities at 240-260-280 nm were in the order of 1: 2: 1, respectively. The concentration of the nucleic acid was determined assu~ng an E$?a,, = 25. The concentration

TMV-RNA

SYNTHESIS

of the nucleic acid was then adjusted to 40 pg/O.l ml. Preparation of polyacrylumide gels and electrophoretic separation of nucleic acid species. The 2.4 % polyacrylamide gels were prepared essentially as described by Bishop et al. (1967). After polymerization the gels were transferred to electrophoresis buffer containing 0.2 % recrystallized SDS (100 ml/ gel) and allowed to stand at 4” for 72 hr prior to use. The gels were then transferred to Plexiglas tubing, one end of which was covered with dialysis membrane. The gels were prerun for 30 min prior to use. Twenty micrograms of nucleic acid in a volume of 0.05 ml electrophoresis buffer containing 5 % sucrose was then applied to the gel and electrophoresis was carried out for either 90 min or 150 min at 10 V/cm, 5 mA/gel. Determination of relative RNA concentration and label distribution in the gels. Immediately after electrophoresis the gels were scanned in a Gilford gel scanner (260 nm) ; and the electropherograms were recorded. The area under each optical density peak was then determined and converted to micrograms of RNA assuming an E%7”nm = 25. After scanning, the gels were frozen in dry ice and sliced into l-mm sections. The gel sections were placed in scintillation vials and dried at 160’ for 12 hr and then swelled with 0.5 ml of a solution made up of 20 % NCS solubilizer, 3.75 % water and 76.25 % toluene for 12 hr at 37” (Haslam et al., 1970). Ten milliliters of toluene-based POPOP-PPO solution was added and the preparations were placed in the dark at 4’ for 12 hr. Radioactivity was then determined in a Packard Tri-Carb liquid scintillation counter. Extraction and density gradient centrijugation of complete TMV. Complete TMV was extracted from infected tissue culture cells after repeated freezing and thawing of the material. Tissue (1.5 g) was ground in a glass homogenizer containing 2.5 ml of extraction buffer (20 mM KCl, 5 mM Mg (C2H30&, 0.1 M Tris. HCl, pH 7.8, 1 mM Cleland’s reagent, 0.25 M sucrose). After homogeniza-

IN TISSUE

CULTURE

789

tion the extract was filtered through “Mira Cloth” to remove cell wall debris The extract was subjected to centrifugation at 33,600 g for 15 min. The supernatant was then centrifuged at 134,000 g for 90 min. The resulting pellet was suspended in 1 ml of extraction medium minus sucrose. This material was layered on a linear 1040 % sucrose gradient (20 mM KCl, 5 mM Mg (C2H30J2, 0.1 M Tris. HCl, pH 7.8). The gradients were subjected to centrifugation at 80,000 g for 90 min in a SW 25.1 rotor and fractionated with an ISCO Model D fractionator coupled to a Model UA-2 U V analyzer (254 nm). The material corresponding to the viral band region of the gradient was collected. After addition of 0.5 mg bovine serum albumin (BSA) the material was precipitated with 10 % trichloroacetic acid (TCA), collected by centrifugation, and washed twice with 5 % TCA. The precipitate was then suspended in 5 ml of distilled water, brought to dryness, and solubilized as described above. Radioactivity was determined in a liquid scintillation counter. The concentration of virus in the virus band was determined assuming an E%%,,, = 3.06. Preparation and use of uridine-3H labeled TMV for marker RNA. Tissue culture cells were inoculated, transferred to filter paper, and incubated in 2 ml of medium containing uridine-3H (25 pCi/ml). The medium was removed and fresh medium added at 24-hr intervals. After 120 hr incubation the cells were harvested and washed with cold 0.25 M sucrose and frozen. The cells were ground and the virus isolated as previously described by Murakishi et al. (1971). Virus to be used as a source of marker RNA was further purified by centrifugation on a 1040% sucrose gradient; material banding in the TMV region was collected by ISCO fractionation. Virus purified in this manner had a specific activity of 288 cpm/pg. To identify the position of viral RNA in the polyacrylamide gels, labeled virus was mixed with unlabeled tissue culture cells 60 hr post inoculation. Total nucleic acids were

790

PELCHER,

MURAKISHI,

then extracted and subjected to electrophoresis for 90 min. The optical density profile and radioactivity was determined as described above. RESULTS

Gel Electrophoresis of Nucleic Acids Extracted from Healthy and T&IV-Infected Tobacco Tissue Culture Cells and IdentiJication of T&W-RNA At 60 hr post inoculation (p. i.) total nucleic acids were extracted from both healthy and TMV-infected tissue culture cells. Electrophoresis of nucleic acids from control tissue culture yielded four discrete optical density peaks (Fig. 1A). The peaks corresponded to DNA (2.5-3.7 mm), 25 S RNA (16.3-22.5 mm), 18 S RNA (27.5-33.7

AND

HARTMANN

mm), and 44 S RNA (57.5-67.5 mm) (Loening and Ingle, 1967). A small optical density peak which migrated slightly faster than the DNA was occasionally observed in electropherograms of nucleic acids from both healthy and infected cells. Its nature or origin was not determined. Nucleic acids extract’ed from TMV-infected tissue culture exhibited the four optical density peaks observed with nucleic acids extracted from healthy tissue plus a fifth peak (7.5-12.5 mm) between the DNA and 25 S RNA (Fig. 1B). This peak was identified as TMV-RNA by coelectrophoresis of unlabeled nucleic acids isolated from cells infected for 60 hr and uridine-3H-TMV-RNA isolated from labeled TMV prepared as described in the Materials and Methods. The coincidence of I

I

I

B

A

1.00

10

0.75

7.5 f,

I 0 F

x

: ; z Y 0 ;

z " 0.50

5.0

0.15

1.5

-=

” ; ;

,

JO

CRACT

ION

40

50

60

FIG. 1. Polyacrylamide gel electrophoresis of nucleic acids extracted 60 hr post inoculation. Twenty micrograms of phenol-extracted nucleic acid was applied to 2.4’$‘, polyacrylamide gels and subjected to electrophoresis for 90 min at 5.0 mA/gel, 90 V/gel. (A) Unlabeled nucleic acids extracted from control cells 60 hr after inoculation with buffer only. Based on relative migration rates the major optical density peaks represent, from left to right, DNA, 25 S RNA, 18 S RNA, and 4-5 S RNA (Loening and Ingle, 1967). (B) Unlabeled nucleic acids extracted from virus-infected cells 60 hr after inoculation. Immediately prior to extraction of the nucleic acids, purified uridine-3H labeled TMV was added to the infected cells to serve as a radioactive marker for TMV-RNA synthesized in the infected cells. After electrophoresis the gels were scanned and sliced into l-mm sections and radioactivity determined. TMV-RNA migrated between the DNA and 25 S host RNA. (---, OD; O-0, cpm).

TMV-RNA

SYNTHESIS

the optical density peak and the radioactivity peak shows that the new peak observed with nucleic acids from infected tissue culture is TMV-RNA (Fig. 1B). Hirai and Wildman (1969) and Fraser (1969) have demonstrated that gel electrophoresis of nucleic acids isolated from tobacco leaf tissue resolves four ribosomal

A

B I

IN TISSUE

CULTURE

791

RNA species. It was reported that two of these RNA species, namely the 25 S and 18 S species, correspond to the subunit RNAs of the 80 S cytoplasmic ribosomes. The 23 S and 16 S RNAs correspond to the subunit RNA of 70 S chloroplast ribosomes. In Fig. 1 it can be seen that no optical density peaks occurred in the area that would correspond

C

B E

A FIG. 2. Optical density profile and radioactivity (circles) of nucleic acids extracted from control and virus-infected cells. The cells were exposed to uridine-SH (25 &X/ml) for 12 hr prior to nucleic acid extraction. Electrophoresis was carried out on 2.4% gels for 150 min at 5.0 mA/gel. Radioactivity determinations were confined to the first 20 mm of the gels, that portion of the gel known to contain TMVRNA. (A) Nucleic acids from control cells labeled for 12 hr; (B) nucleic acids from infected cells labeled 12-24 hr post inoculation (p.i.); (C) nucleic acids from infected cellslabeled36-48 hr p.i.; (D) nucleic acids from infected cells labeled 48-60 hr p.i.; (E) nucleic acids from infected cells labeled 72-84 hr p.i.; (F) nucleic acids from infected cells labeled 132-144 hr p.i.

792

PELCHER,

MURAKISHI,

to the 23 S and 16 S chloroplast ribosomal subunit RNAs. This suggests that either the chloropl~t ribosome content of tissue culture cells is extremely low or the extraction technique employed was not effectively extracting chloroplast ribosomal RNA. In an attempt to resolve this problem, total nucleic acids were extracted from both healthy and TMV-infected leaf tissue of H-38 tobacco plants. Although the results are not depicted here, both 80 S and 70 S ribosomal subunit RNAs were extracted from leaf tissue and separated by gel electrophoresis.

AND HARTMANN

0

Culture Cells At 12-hr intervals after inoculation, cells were transferred to liquid medium containing u~dine-3H (25 $&‘ml) and incubate for 12 hr. At the end of each labeling period, total nucleic acids were extracted from the cells and subjected to electrophoresis for 2.5 hr. Figures 2A and 2B are optical density profiles of nucleic acids extracted from uninfected cells and from cells infected for 24 hr, respectively. A small optical density peak corresponding in position to TMV-RNA is present in the electropherogram of nucleic acids extracted from infected cells 24 hr p.i. During the 12-24-hr labeling period only a small amount of uridine-3H was incorporated into TMV-RNA (Figs. 2B, and 3). Between 3648 hr p.i. the rate of viral RNA synthesis as expressed by uridineJH incorporation had increased to approximately 4 times that of the 12-24 hr p. i. period (Figs. 2C and 3). The amount of viral RNA present in the infected cells doubled during the second 24hr period following inoculation (Fig. 3). During the 48-60 hr p. i. period the rate of incorporation of uridine-3H into viral RNA reached its peak, appro~mately 6 times that of the 12-24 hr p. i. period (Figs. 2D and 3). During the first 60 hr p.i. the rate of uridine3H incorporation into viral RNA and the rate of accumulation of viral RNA was nearly linear (Fig. 3). After reaching a peak between 48 and 60 hr

24 HOURS

48

72 POST

96

120

144

INOCULATION

FIG. 3. Rate of incorporation of uridine-3H into viral RNA (O---O) and accumulation of viral RNA (@-0). Labeling, extraction, and electrophoresis of the nucleic acids was carried out as described in Fig. 2. Rate calculations were made by determining cpm/min/gel corresponding in position to the TMV-RNA optical density peak. All calculstions were corrected for background radioactivity observed with nucleic acids from control cells. Total viral RNA determinations were made by converting the area under the viral RNA optical density peak to micrograms RNA sssuming an &X% = 25. Both rate and total viral RNA determinrstions represent the average of two experiments.

p. i. the rate of uridine-3H incorporation into viral RNA began to decline. By the 60-72 hr period, the rate of uridine-3H incorporation into viral RNA had decreased to approximately 60% that of the 48--60-hr period. During the 72-84 hr p.i. period the rate of uridineJH incorporation appeared to increase slightly over the 60-72-hr period (Figs. 2E and 3). The apparent increase in rate over the 60-72-hr period was observed in duplicate expe~ments; however its significance is not known. During the 132-144 hr p.i. period incorporation of uridine3H into viral RNA was only 13 % that of the peak rate (Figs. 2F and 3). While the rate of uridine-3H incorporation was decreasing (SO-168 hr p.i.) the amount of viral RNA in

TMV-RNA

SYNTHESIS

the infected cells remained relatively constant (Fig. 3). To determine whether the de&e in the rate of incorporation of uridine-3H into viral RNA after 60 hr p.i. was due to a change in the cellular pool of nucleic acid precursors, the specific activity (cpm/rg) of the cellular RNA wa monitored at 24hr interva&s following ~n~~ulatio~. This was a~~~rn~hshed by determining the specific activity of the 25 S host ribosomal component. The area under the 25 S RNA peak wa8 determined by t~angu~ation and converted to total, optical density units and the ~~rogran~ of RNA present were calculated assuming an Ei$nm = 25. The specific activity of the 25 S RNA remained relatively constant throughout the 16%hr period studied (Table 11, sugges~ng that the decline in the rate of uridine-?I jn~or~orat~on into viral RSA was not due to a change in the cellular pool of nucleic acid precursors. At 12-hr intervals after inoculation, the specific activity of the viral RNA was st1s0 determined. During the first 60 hr pi. the specific activity of the viral RNA increased, reaching 8.5 X JO” cpm/pg between 48 and 60 hr p.i., after whioh time the specific activity declined to 1.1 X lo3 cpm/pg by 168 hr p.i. (Table 1). The speeifie activity of the viral RBA increased slightly during the 7% 84 hr pi. period; this corresponds to the slight apparent increase in rate of synthesis observed during this period.

The relationship between viral RNA synthesis and the formation of complete virus was studied to further characterize the events of virus replication following infection of tissue culture cells with T&IV, Aliquots of infected cells (1.5 g), comparable to those used for total nucleic acid isolation, were used to follow the kinetics of formation of complete virus. The cells were repeatedly frozen and thawed to destroy eelMar ribosamai material. The ceffs were then ground,

IN TISSUE

793

CULTURE TABLE

Labeling period (hr p.i.) 12-24 24-36 36-43 4&60 6&72 72-84 84-96 108-120 132-144 156-163

I

@m/&g RNA Cellular 25 Sa 3379 4171 2976 3770 4779 3597

..I.

Viral” 4966 6305 8095 8539 4393 5206 36II 2548 1023 1111

_I-. a Specific activity of 25 S host ribosomal RNA WBB determined at 24-hr intervals after inoculation. Labeling, extraction, and electrophoresis wn.~ carried out w in Fig. 2, except that radioactivity determinations were made throughout the gei. The sea under the 25 S RNA optical density peakwas converted to micrograms of RNA. Total radioactivity corresponding to the 25 S optical density peak WBBdivided by the total amount of 26 S RNA to give the specific activity (cpm/pg 25 S RNA). 6 Specific activity of the viral RNA was determined at 12-hr intervals after inoculation. The specific activity of the viral RNA wm determined aa described for the 25 S host riboaomal RNA. Values represent the average of two experiments.

and complete virus was isolated as described in ~~ate~als and Methods. The pelleted virus was then suspended in 1 ml of extraction medium and subjected to sucrose density gradient centrifugation for 90 min. The gradients were then fractionated by means of an lSCU density gradient analyzer. The material corresponding to the virus band was oollected and precipitated with TCA and the radioactivity was determined. The incorporation of in viva synthesized uridine-“H labeled T&IV-RNA into complete virus is depicted in Fig. 4. Tha amount of u&&e3H-T&W-RNA incorporated into complete virus during the 12-hr labeling periods increased rapidly from 24 to 60 hr p.i. After the first 60-hr period the rate of incorporation of uridine-%I labeled RNA into complete virus declined ~hrougho~~tthe rest of

794

PELCHER,

MURAKISHI,

26. 24 f 22 % 20 D 18. In ?; 16. 0 c

14.

x

12.

E L ”

lo-

I 9-0

x

8 64-2

2 24 HOURS

48

72 POST

96

120

144

INOCULATION

FIG. 4. Rate of incorpoiation of in tivo synthesized uridine-3H TMV-RNA into complete virus (0-O) and accumulation of complete virus (e---0). At 12-hr intervals after inoculation, cells were exposed to uridine-3H (25 &i/ml) for a 12-hr period. Complete virus was extracted as described in Materials and Methods and subjected to centrifugation on 1040% sucrose gradients at 80,000 q for 90 min. Gradients were fractionated with an ISCO fractionator coupled to a U.V. analyzer (254 nm) . The viral band region was collected, precipitated with 10% TCA, and washed with 5% TCA, then radioactivity was determined. The TMV concentration was calculated by converting the area under the virus optical density peak to micrograms complete virus assuming an

E,$?, = 3.06. the 168hr period studied. The accumulation of complete virus continued until approximately 96 hr p.i. after which time there was little detectable change in the amount of complete TMV (Fig. 4). By 120 hr p.i. there was an apparent loss of virus. The rate of accumulation of complete TMV increased in a nearly linear manner between 24-60 hr p.i. (Fig. 4). Although accumulation of complete virus continued after 60 hr p.i., the rate of accumulation declined rapidly. Observations on Crystalline Formation in Inoculated

Viral Inclusion Tissue Culture

As previously reported (Murakishi et al., 1970), tobacco tissue culture cells inoculated

AND

HARTMANN

with TMV produce crystalline viral inclusions. Such inclusions indicate that a high virus titer was reached in inclusion-bearing cells (Murakishi et al., 1970). During the course of the experiments reported here an attempt was made to correlate the appearance of crystalline viral inclusions with viral RNA synthesis. At 12-hr intervals after inoculation random clumps of cells were observed microscopically at 100 and 430 X magnifications to detect viral inclusions. The earliest that crystals were observed was 44 hr p.i. In such cases only a single or very few crystals were present; however, by 72 hr p.i. large aggregates of inclusion-bearing cells could be observed. The number of cells in individual aggregates varied from 5 to 25 cells per aggregate. By 72 hr p.i. virtually all cells of such aggregates contained viral crystals. The number of such inclusion-bearing cell aggregates varied widely from experiment to experiment. DISCUSSION

During the first 60 hr after inoculation of tissue culture cells, the rate of viral RNA synthesis increased, reaching a peak during the 48-60 hr period (Fig. 3). This conclusion is based on the observation that both the rate of uridine-3H incorporation into viral RNA and the specific activity of the viral RNA increased during each 12-hr labeling period up to 60 hr p.i. The increasing specific activity of the viral RNA (Table 1) indicates that during each successive 12-hr period, increasing amounts of viral RNA were synthesized, the largest amount being synthesized during the 48-60-hr period. At this time the specific activity of the viral RNA reached 8.5 X lo3 cpm/pg. During the first 60 hr the amount of viral RNA increased in a nearly linear manner (Fig. 3). After the first 60 hr, the rate of uridine-3H incorporation into, and the specific activity of, the viral RKA declined (So-168 hr p.i.), suggesting a continual decline in the rate of synthesis. There was a slight increase in the rate of uridine-3H incorporation into viral

TMV-RNA

SYNTHESIS

RNA during the 72-84 hr period (Fig. 3). A possible explanation for this increase will be discussed later. That the decline in uridine3H incorporation into viral RNA was not due to a drastic change in the cellular nucleic acid precursor pool is indicated in Table 1. The specific activity of the 25 S host ribosomal RNA component remained relatively constant throughout the 16%hr period studied. If there was a change in the precursor pool it’ would have been reflected in the amount of uridine-3H incorporated into this ribosomal component. That a decline in the rate of viral RNA synthesis occurs after the first 60 hr is further supported by the fact that there is little increase in the amount of viral nucleic acid after this time (Fig. 3). Observations on the accumulation of complete virus suggests that viral protein synthesis closely parallels viral RNA synthesis in infected cells. The peak rate of incorporation of in viva synthesized uridine-3H-TlVIVRNA into complete virus and t’he peak rate of accumulation of complete virus (Fig. 4) occurred during the 48-60-hr period, the period of most, rapid viral RNA synthesis (Fig. 3). This observation suggests that viral RNA is incorporated into complete virus rather quickly after its synthesis. The close correlation between the decline in the rate of accumulation of complete virus (Fig. 4) and the decline in the rate of viral RNA synthesis (Fig. 3)-observations made by independent techniques-confirms the contention that viral synthesis is declining during the 60-168 hr p.i. period. Bassanis et al. (1958) have shown that virus spread in inoculated tobacco callus was very slow; therefore, the rapid increase in the rate of virus synthesis observed during the first 60 hr p.i. apparently is not due to extensive virus spread. The tissue culture used in this study was composed of numerous small cell aggregates. Recent electron microscopic observations indicate that the cells of such aggregates arc connected by plasmodesmata (Hartmann, manuscript in preparation). Plasmodesmata could allow for rapid virus

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795

spread throughout each inoculated aggregate. This would lead to or be concurrent with rapid virus synthesis, ultimately leading to crystalline viral inclusion formation in all the cells of the aggregate, after which time the rate of virus synthesis would decline. Our observations on viral RNA synthesis, accumulation of complete virus and crystalline viral inclusion formation and distribution are consistent with this idea. At 44 hr picrystals were rarely observed; however, by 72 hr p.i. large aggregates of crystal-bearing cells were present. This period (44-72 hr p.i.) corresponds closely to t’he period of most rapid viral RNA synthesis and complete virus accumulation (48-60 hr p.i.). After 72. hr p.i. there was little or no increase in the number of aggregates containing crystals. Aggregates of cells lying in close contact with crystal-bearing aggregates appeared to remain free of crystalline inclusions as late as 168 hr after inoculation. The work of Hildebrandt and Riker (1958) and Hirth and Lebeurier (1965) also indicate that inoculated callus consists of aggregates of infected cells enmeshed in a network of apparently healthy cells. Hansen and Hildebrandt (1966) have demonstrated that virus-infected callus cells are still capable of cell division. It is possible that the slight increase in the rate of viral RNA synthesis (Fig. 3) and the increase in the rate of accumulation of complete virus (Fig. 4) observed during the 72-S4-hr period could be due to a second round of virus synthesis following cell division. After the initial rapid spread of the virus throughout the cells of inoculated aggregates, it is reasonable to assume that further spread of the virus is limited and probably occurs only through division of infected cells. ACKNOWLEDGMENTS The authors wish to thank Elliot Light for his technical assistance during preliminary stages of this work. Appreciation is also expressed to Drs. A. H. Ellingboe, G. R. Hooper, and L. F. Velicer for reviewing the manuscript. Journal Paper Number 5671 of the Michigan Agricultural Experiment Station.

796

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MURAKISHI,

REFERENCES BABOS, P. (1969). Rapidly labeled RNA associated with ribosomes of tobacco leaves infected with tobacco mosaic virus. ~~ro~og~39, 893-960. BEACEY, R. N., and MURAKISHI, H. H. (1971), Local lesion formation in tobacco tissue culture. Phytopathology 61, 877-878. BEACHY, R. N., and MURA~ISHI, H. H. (1971). Proteins from cultured cells of Xanthi-nc tobacco inoculated with tobacco mosaic virus. Phytopathology 61, 848 (Abstract). BISHOP, D. H. L., CLAYBROOK, J. R., and SPIEQELMAN, S. (1967). Eleotrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26,373-387. DIENER, T. 0. (1962). Isolation of an infectious, ribonuole~e-sensitive fraction from tobacco leaves recently inoculated with tobacco mosaic virus. ViTology 16, 140-146. FRASER, R. S. S. (1969). Effects of two TMV strains on the synthesis and stability of ehloroplast ribosomal RNA in tobacco leaves. Mol. Gen. Genet.106,73-79. HANSEN, A. J., and HILDEBRANDT, A. C. (1966). The distribution of tobacco mosaic virus in plant callus cultures. V~~oZogy28, 15-21. HASLAM, E. A., HAMPSON, A. W., EGAN, J. A., and WHITE, D. 0. (1970). The polypeptides of influenza virus. II. Interpretation of polyaorylamide gel electrophoresis patterns. ViroEogy 42,~565. HILDEBRANDT, A. C., and RIKER, A. J. (1958). Viruses and single cell clones in plant tissue culture. Fed. Pro&. Amer. Sot. Exp. Biol. 17, 986-993 f HIRAI, A., and WILDMAN, S. G. (1967). Intracellu-

lar site of assembly of TMV-RNA and protein. Virology 33,467-473. HIRAI, A., and WILDMAN, S. G. (1969). Effect of TMV multiplication on RNA and protein synthesis in tobacco chloroplasts. Virology 38, 73-82. HIRTH, L., and LEBEURIER,

sur la sensibilite

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