VIROLOGY 3,554-559 (1957)

The Early Events of Infection With Tobacco Mosaic

Virus Nucleic Acid’

ALBERT SIEGEL, WILLIAM GINOZS, AND SAM G. WILDMAN

Botany Department and Atomic Energy Project, University of California, Los Angeles

Accepted March 5, 1967

The early events of infection with strains Ul and U2 of tobacco mosaic virus, and the nucleic acids isolated therefrom, were compared in the local lesion host Nicotiana glutinosa. The technique used was to irradiate infective centers with ultraviolet light at different times after initiation of infection. Infective centers initiated by intact virus display an initial lag period during which no change in resistance to ultraviolet light occurs. This lag period is 2js hours for the U2 strain and 5 hours for the Ul strain. In contrast, infec- tive centers caused by the nucleic acid isolated from these two strains behave alike and display little or no lag before they begin to increase in resistance to ultraviolet light. The conclusion is reached that when infection is initiated with the intact virus, the nucleic acid moiety of the virus is freed from the protein moiety soon after infection is established and before virus replication begins.

ZNTRODUCTION

It has been demonstrated that nucleic acid comprises the genetic ma- terial of certain bacteriophages (Hershey and Chase, 1952) and of tobacco mosaic virus (TMV) (Gierer and Schramm, 1956; Fraenkel-Conrat, 1956). Since it is possible to initiate infection with the nucleic acid moiety of TMV, this system offers the unique opportunity of comparing the infection initiated by nucleic acid with that caused by the intact virus. We have previously studied the early events of the intact virus infec- tion by the t#echnique of measuring the ultraviolet light (UV) sensitivity of infective centers at different times after inoculation (Siegel and Wild-

1 This work was supported in part by a research grant, E536, from the Na- tional Institutes of Health, U. S. Public Health Service, and Contract AT(ll-l)- 34, Project 8 of the Division of Biology and Medicine, U. 8. Atomic Energy Com- mission.

554

INFECTION WITH TMV NUCLEIC ACID 555

man, 1956). This communication presents the results of similar experi- ments in which nucleic acid was used to initiate infection.

MATERIALS AND METHODS

Infectious nucleic acid was prepared from TMV strains Ul and U2 (Siegel and Wildman, 1954) essentially by the heat-detergent method of Fraenkel-Conrat and Williams (1955). For the Ul strain 8 minutes of heating at 50” at pH 8.8 in 1% sodium dodecyl sulfate are required to free the nucleic acid from the protein moiety of the virus. In the case of U2, only 5 minutes are required. In all cases, to remove possible virus con- tamination, two centrifugations for 1 hour each at 40,000 rpm in rotor no. 40 in the Spinco centrifuge, model L, followed 2 or 3 cycles of the pre- scribed alcohol precipitation. The final nucleic acid preparations had the following UV absorption characteristics: maximum at 258 rnp, minimum at 230 rnk, maximum/minimum ratio 2.7 to 3.0, and a 2601280 ratio of 2.0. Of the numerous preparations tested, the infectivity of all was completely inactivated with ribonuclease treatment under conditions where comparable control samples of virus were virtually unaffected. The infectivity of ten of the nucleic acid preparations was compared with that of the parent virus preparations. They had 0.05 to 0.23 % (average, 0.1%) of t,he infectivity of an equal amount of nucleic acid contained in the parent virus preparations. This figure is in agreement with that of Gierer and Schramm (1956), whose procedure involved the use of water- saturated phenol for the separation of the nucleic acid from the protein.

Irradiation of nucleic acid and virus as well as of infected leaves of Nicotiuna glutinosa was carried out as previously described (Siegel and Wildman, 1956). Leaves were maintained at 20” between the time of in- oculation and irradiation.

EXPERIMENTAL RESULTS

We have previously reported that the TMV strains Ul and U2 differ 5x-fold in sensitivity to inactivation with UV (Siegel and Wildman, 1956). The nucleic acids isolated from these strains, however, both have a sensitivity identical to that of the more sensitive U2 strain (Siegel et al., 1956). In addition, we have demonstrated that infective centers initiated by the two virus strains have initial UV sensitivities which are similar to that of the virus strains irradiated in vitro. Figure 1 summarizes this information and also presents the additional information that nucleic acid infective centers have t’he same initial UV sensitivity as that of

556 SIEGEL, GINOZA, AND WILDMAN

UV Dose (minutes> FIG. 1. A comparison of UV survival of nucleic acid irradiated in vitro and

nucleic acid infective centers irradiated immediately after inoculation. KEY: 0, Ul nucleic acid; 0, Ul nucleic acid infective centers; q , U2 nucleic

acid; n , U2 nucleic acid infective centers; ----, U2; - - - -, U2 infective cen- ters; . . . ., Ul and Ul infective centers (Siegel and Wildman, 1956).

nucleic acid irradiated in vitro. The Ul nucleic acid infective center be- haves differently from that induced by the intact Ul virus, in that the former is considerably more sensitive to UV inactivation than is the latter. The same also seems to be true in the case of U2, but here the difference in sensitivity of nucleic acid and intact virus infective centers is not as great.

In order to compare more effectively infection by the nucleic acid with that by the intact virus, the change in sensitivity of the infective center to UV was followed as a function of time after inoculation. Experiments such as these have already been reported for the infection initiated with the intact U2 strain (Siegel and Wildman, 1956). The results of similar experiments with the Ul strain and with the nucleic acid of the two strains are presented in Fig. 2. When the infectious agent is the intact virus, there is a period after inoculation during which no change in UV

1.0

CJ, c .- > 0.5

.a > L

G

0.2 : .- + 0 0

Lt 0.1

INFECTION WITH TMV NUCLEIC ACID 557

0 2 4 6 6 10 12 14

Hours After Inoculation FIG. 2. Ultraviolet light survival of infective centers as a function of time after

inoculation. Dose of UV: intact U2 and nucleic acid, 90 seconds; Ul, 5 minutes. KEY: 0, Ul nucleic acid; El, U2 nucleic acid; v, intact U2; 0 and X, intact Ul.

sensitivity occurs. This lag period is approximately 2% hours for the U2 strain and somewhat over 5 hours for the Ul strain. In contrast, when nucleic acid is the infecting agent, two marked differences are apparent: (a) the lag period is greatly reduced or missing entirely, and (b) iden- tical behavior is exhibited by the nucleic acid infective centers of both strains.

The increase in survival against UV with time, shown by the infective centers induced by the U2 strain, occurs in two phases (Siegel and Wild- man, 1956). During the first phase there is a decrease in slope of the initial exponential survival curve. The second phase, starting at the 5th hour after inoculation, consists of a change in the shape of the survival curve from exponential to multitarget in nature. The infective centers induced by nucleic acid also exhibit these two phases, although the shift from the exponential to the multitarget curve occurs sooner, between the 2nd and 3rd hour after inoculation. Preliminary data indicate that the

558 SIEGEL, GINOZA, AND WILDMAN

change in the shape of the survival curve occurs when the infective centers of nucleic acid and U2 attain the same resistance to UV. We have suggested that the shift from the exponential to the multitarget curve indicates the time when virus replication begins (Siegel and Wild- man, 1956). On the basis of this interpretation, virus replication is pre- ceded by a preparatory stage involving an increase of resistance to UV by the infective centers of both nucleic acid and intact virus.

DISCUSSIOK

The data presented in this paper demonstrate that the TMV nucleic acid preparations behave differently from the intact virus both in their sensitivity to inactivation with UV and in their biological behavior. This provides evidence, additional to that obtained by chemical means (Fraenkel-Conrat, 1956; Gierer and Schramm, 1956), that the infectious entities present in the nucleic acid preparations cannot be contaminant intact virus particles. The conclusion is thereby fortified that the nucleic acid moiety of the TMV particle is in itself capable of initiating the in- fection and thus, is the bearer of the total genetic information of the virus particle.

The UV sensitivity of the infective center initiated by nucleic acid be- gins to decrease very soon after initiation, in contrast to a lag of several hours exhibited by the TMV-induced infective center. This indicates that the initial events of TMV infection are concerned with the release of the nucleic acid from the protein moiety of the virus particles.

The Ul strain has a considerably longer lag than does the U2 strain. One possible explanation for this phenomenon is that the nucleic acid is released with greater difficulty from the Ul particles. There are several lines of independent evidence for a more intimate relationship between nucleic acid and protein in strain Ul than in strain U2 : (1) The combina- tion of the nucleic acid and protein in the Ul strain is such as to afford appreciable protection to the nucleic acid from the damaging effects of UV (Siegel et al., 1956). (2) The nucleic acid of the Ul strain is released with greater difficulty in vitro than that of the U2 strain by the heat- detergent method employed in its isolation. (3) The Ul strain is more re- sistant to heat denaturation than is the U2 strain (Siegel et al., 1956). Thus, the greater affinity between nucleic acid and protein in the Ul strain, as revealed by physical and chemical studies of the extracellular rod, may also be reflected in the increased time to complete a biological process.

INFECTION WITH TMV NUCLEIC ACID 559

Both the infective centers initiated by nucleic acid and the U2 virus undergo an increase in resistance to UV before virus replication begins. This initial rise in resistance may represent the movement of the infect- ing agent from the surface to the inside of the cell, where the cell may contain protective substances which shield the infecting agent from the damaging effects of UV. One consequence of this model is that the nucleic acid starts its movement almost immediately after contact with the cell whereas the intact virus does not. A lag of approximately 2% hours occurs before the U2 infective center begins to increase in resistance. We suggest that nucleic acid is freed from the protein moiety of the virus during this lag period. The site of “protein stripping” in this case would be close to the surface of the leaf since the resistance to UV during the lag period is little changed from that of the extracellular virus particle.

On the other hand, the amount of UV-absorbing materials of the cell may be insufficient to shield the infecting agent from UV damage. In this case the resistance increase may result from a combination of the infecting nucleic acid and a host protein in a manner similar to that existent in the intact Ul virus.[It will be recalled that the type of nucleic acid-protein binding found in the intact Ul virus leads to protection of the nucleic acid from the damaging effects of UV (Siegel et al., 1956).] On the basis of this model, the nucleic acid is released from the intact U2 virus during the initial lag period. Subsequent to release from its orig- inal protein envelope, the nucleic acid is bound to a host protein and attains the observed resistance to UV.

REFERENCES

FRAENKEL-CONRAT, H. (1956). The role of the nucleic acid in the reconstitution of active tobacco mosaic virus. J. Am. Chem. Sot. 78, 882-883.

FRAENKEL-CONRAT, H., and WILLIAMS, R. C. (1955). Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc. Natl. Acad. Sci. U. S. 41, 690-698.

GIERER, A., and SCHRAMM, G. (1956). Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703.

HERSHEY, A. D., and CHASE, M. (1952). Independent function of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39-56.

SIEGEL, A., and WILDMAN, S. G. (1954). Some natural relationships among strains of tobacco mosaic virus. Phytopathology 44, 277-282.

SIEGEL, A., and WILDMAN, S. G. (1956). The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light. Virology 2, 69-82.

SIEGEL, A., WILDMAN, S. G., and GINOZA, W. (1956). Sensitivity to ultraviolet light of infectious tobacco mosaic virus nucleic acid. Nature 178, 1117-1118.