VIROLOGY 1, 165-175 (1955)

Infectivity of Turnip Yellow Mosaic Virus Containing 8-Azaguanine

R. E. F. MATTHEWS

Agricultural Research Council Plant Virus Research Unit, Molteno Institute, Cambridge, England

Received December Y, 1954

SUMMARY

Treatment with 8-azaguanine delays the development of turnip yellow mosaic virus m-hen small Chinese cabbage plants are used. Small amounts of 8.azaguanylic acid were found in the nucleic acid of virus from treated plants. The infectivity of virus preparations containing S-azaguanine was less than that of normal preparations containing the same amount of virus nucleic acid. No difference in the proportion of protein and nucleoprotein particles was detected in virus preparat,ions from control and from treated plants.

A method is described for estimating virus protein in clarified plant sap. This method is based on time for precipitation with antiserum and gives more precise estimates than either optimal proportions or end-point de- terminations.

INTRODUCTION

The guanine analog, S-azaguanine, inhibits growth of a variety of organisms. In all cases that have been adequately examined bhis in- hibition is accompanied by an incorporation of 8-azaguanine in the ribonucleic acid (RNA). It has generally been considered that anti- metabolites act by blocking enzymes involved in the utilization of the metabolite. However, the fact that 8-azaguanine is incorporated into RNA suggests that the compound may have its effect not by blocking enzymes involved in the utilization of guanine but by causing non- functional RNA to be produced. In most of the systems that have been studied (e.g., bacteria, mammalian tumor and liver tissue) it would be difficult to demonstrate that the RNA containing 8-azaguanine was nonfunctional. In bacterial viruses there is direct experimental evidence indicating t,hat deoxyribonucleic acid (DKA) may be the genetic ma- t,erial of the virus (Hershey and Chase, 1952). All plant viruses that

165

166 R. E. F. MATTHEWS

have been adequately examined have been found to contain only RNA and protein. Although there is no direct evidence, it seems probable by analogy with the bacterial viruses that RNA is the genetic material of plant viruses. This view receives support from the example of turnip yellow mosaic virus. In plants infected with this virus two types of particle are present: a nucleoprotein which is infectious, and an ap- parently identical protein without nucleic acid, which is not infectious (Markham and Smith (1949)).

Thus with the plant viruses it should be possible to detect the presence of biologically ineffect’ive RNA by comparing the infectivity of 8-aza- guanine-containing virus preparations with control preparations. With tobacco mosaic virus, incorporation of 8-azaguanine appeared to reduce the number of local lesions obtained per unit weight of virus nucleic acid (Matthews, 1954a). However, with this virus the degree of end-to- end aggregat’ion of the rod-shaped particles could influence infectivity even in tests with freshly expressed crude sap; and such aggregation rules out infectivity experiments with purified virus preparations. For this reason one of the spherical plant viruses would be much more suitable for this type of experiment. In our early tests with turnip yellow mosaic virus (TYMV), in which fairly large Chinese cabbage plants were inoculated, 8-azaguanine had no detectable effect on t’he development of the virus (Matthews, 1954b). We found subsequently that, if very small plants are used, 8-azaguanine has an effect on virus production and disease development comparable to that with tobacco mosaic virus. The experiments described below suggest that the incorporation of 8-azaguanine into the nucleic acid of TYMV renders a proportion of the virus particles incapable of initiating infection.

Jeener (1954) has obtained evidence that the virus protein found in TYMV infections is a precursor of the complete nucleic acid-containing virus particle. We have estimated the ratio of virus protein and nucleo- protein in purified virus preparations and in clarified sap from 8-aza- guanine-treated plants and from control plants. Treatment with 8-aza- guanine caused no detectable change in the proportion of the two kinds of particle.

MATERIALS AND METHODS

Plants and Treatments

Chinese cabbage plants grown in potting mixture were inoculated with the type strain of turnip yellow mosaic virus when several leaves

8-AZhGUAR’INE AND VIRUS INFECTIVITY 167

were 3 to 5 inches long. Concentrations of 8-azaguanine between 0.005 and 0.02 M in solution in 0.1% KaHC03 were sprayed on the leaves every 2 or 3 days. Treatments were begun either 2 or 3 days before inoculation or the day after inoculation. Control plants were sprayed with about the same total amount of 8-azaguanine in the 24-hour period before harvesting.

Infectivity Measurements

There is no host known for TYMV in which easily countable necrotic local lesions are regularly produced. In Chinese cabbage the local lesion rrsponse varies considerably. Some leaves produce clearly marked chlorotic lesions. Others may produce faint lesions of this t,ype which are difficult to count, and many produce none at all. Necrotic or pigmented lesions occasionally appear. Nevertheless, the response of any one leaf is uniform so that comparisons made on half-leaves are valid.

To compare t,he infectivity of control virus and S-azaguanine-con- t,aining virus, the t,wo preparat(ions were inoculated in a series of three tenfold dilutions into opposite half-leaves. Considerably more leaves were inoculated t,han the number of comparisons desired. All leaves showing any lesions were counted, and those on which both half-leaves contained more than one hundred lesions were discarded. As a check on the proportion of leaves with obscure lesions, all leaves were counted independently by t’wo observers. To determine t)he significance of differ- ences between numbers of local lesions produced by two preparations, a transformation of t,he type x = log,& + c) was used, where y = number of local lesions and c is a constant (Kleczkowski, 1950).

Isolation and Analysis of Virus

The alcohol-ammonium sulfate procedure of Markham and Smith (1949) was used to isolate crystalline virus. Aft’er three crystallizations with ammonium sulfat,e (one-third sat,uration), the optical density at 260 rnp (DZEO) for 0.1 mg/ml of the virus (determined from dry weights) and t,he percentage of phosphorus remain fairiy constant. This number of crystallizations was therefore employed for most preparations. The ahohol method (h!Iarkham and Smith, 1949) was used in most experi- ments for isolating the virus nucleic acid. Base analyses and the isolat’ion of 8-azaguanylic acid were carried out by the methods used for tobacco mosaic virus (Matthews, 1954a). Phosphorus determinations were made by t’he method of Allen (1940).

168 R. E. F. MATTHEWS

Estimation of the Ratio of Virus Protein and Nucleoprotein in Purijied Preparations

Markham and Smith (1949) found that the quantity of the protein component in normal preparations was fairly constant. In attempting to detect small differences in the ratio of virus protein and nucleoprotein the phosphorus content or the optical density at 260 rnp for 0.1 mg/ml of virus was used, the total amount of purified material being deter- mined by dry weights. These two measures are fairly closely correlated. Compared with these two methods, the ratios of optical density 260 mp/240 rnp and 260 mp/280 rnk appear to be relatively insensitive to small changes in composition.

Virus preparations obtained after several ammonium sulfate crystal- lizations can be separated into fractions containing greater and smaller amounts of nucleic acid than the original material. A solution of am- monium sulfate is added to the virus preparation dropwise, with stirring, until the solution just begins to appear turbid. The material which crystallizes on standing contains a greater proportion of nucleic acid than the original preparation. The material remaining in the supernatant fluid, which is a small proportion of the total, crystallizes on the addi- tion of further ammonium sulfate. This material contains less nucleic acid. For example, a preparation having i&o = 0.81 for 0.1 mg/ml was separated into two fractions having DzGO = 0.83 and 0.72 for 0.1 mg/ml. It has not yet been established whether such differences repre- sent a partial fractionation of the virus protein and nucleoprotein or of virus and extraneous material.

Because of the possibility of varying the composition, when isolating virus from the controls and from %azaguanine-treated plants care was taken to use the same concentration of ammonium sulfate for virus crystallization for both preparations.

Estimation of Virus Composition in Heat-Clarified Plant Sap

Since the isolation procedure might alter the composition of the virus obtained, serological methods were used to determine the ratio of virus protein and nucleoprotein in plant sap clarified by heating to 54” for 5 minutes. Estimates of the amount of virus nucleic acid were made by the serological chromatographic procedure used for tobacco mosaic virus (Matthews, 1954a). Because of the relative instability of TYMV at 50” in the presence of serum, incubations with antiserum were made at room temperature for 1 to 2 hours, followed by overnight storage at about 4’. The estimates of the concentration of virus in plant

S-AZAGUANINE AND VIRUS INFECTIVITY 169

sap obtained by this method were higher than those calculated from the yields of purified virus, particularly in early infections where the virus concentration was low. In one experiment, for example, plant sap contained 0.31 mg/ml of virus estimated by the serological chro- matographic procedure, and 0.14 mg/ml from the yield of purified virus. Such differences were largely accounted for by virus remaining in the supernatant fluids after each ammonium sulfate crystallization.

The serological method used in the present work to estimate the amount of virus protein in heat-clarified sap was based on the time taken for specific precipitation with antiserum. There is good evidence that the prot’ein and nucleoprotein components of TYMV react in a very similar if not identical manner with virus antiserum (Markham and Smith, 1949; Jeener, 1954). We can therefore assume that serological estimations of virus by methods using optimal proportions or the virus end point) measure the total amount of virus protein. These methods, however, have an error of about 50%. Much greater accuracy can be obtained by recording the times taken for visible precipit)ation to occur in t,he region of ant’iserum excess, using twofold dilutions of virus and a constant dilut,ion of antiserum. A dilution of antiserum is employed which gives visible precipitation at the cx optimum in about, 1 minute. A 0.5-ml portion of each reagent is mixed in a small tube and placed immediately in a constant-temperature water bath fitted with windows and rear lighting. The time taken for precipitation to become visible with a hand lens is noted. Mixtures giving times between about 1 and 60 minutes are recorded. Duplicate tests are made for each mixture, and t,he mean figure taken. The logarithms of these bimes for precipit,a- t)ion are then plotted against the logarithms of the virus dilution. The points fall approximately on a straight line. For a series of virus prepara- tions a series of lines of the same slope can be drawn in by inspection. The displacement of these lines along the virus axis gives a measure of t’he relative virus concentrat)ion. If the antiserum is standardized with a virus preparat,ion of known protein concentration, then the weight of the virus protein can be determined. Figure 1 illustrates this method.

RESULTS

E$ect of 8-Azaguanine Treatment on Disease Development and virus Multiplication

If young plants are sprayed with S-azaguanine, the development of systemic symptoms of TYMV is delayed by a few days. The yield of virus is reduced if plants are harvested within about 14 days (Table 1).

170 R. E. F. MATTHEWS

1 l/4 l/8 l/16 l/32 l/64

Virus dilution

FIG. 1. Times taken for precipitation. Twofold dilutions of a standard (S) and two experimental (A and B) preparations of TYMV with a constant dilution of antiserum.

TABLE 1

EFFECT OF 8-AZAGUAxINE TREATMENT ON NUMBER OF SYSTEMICALLY INFECTED PLANTS AND YIELD OF VIRUS 14 Days AFTER

INOCULATION

No. of plants systemically

infected

Expt. 1 Control 76/83 0.140 %Azaguanine-treated 40/216 0.0306

Expt. 2 Control 48/48 0.144 8-Azaguanine-treated O/96 0.049

0 Estimated from yields of purified virus.

Composition of the Virus in Control and in 8-Axaguanine-Treated Plants

Nucleic acid composition. 8-Azaguanylic acid was found in the alkaline hydrolyzates of nucleic acid from virus from treated plants but not in those from control plants treated with Sazaguanine just before har- vesting. The size and density of the fluorescent spots obtained by paper elect,rophoresis of the nucleotides at pH 9 suggested that’ the amounts present were less than 1% of the guanylic acid, an amount too small to be estimated accurately by density measurements.

With tobacco mosaic virus, base analyses showed a deficiency of about 3 % in guanine, which was accounted for by the incorporation of a corresponding amount of S-azaguanine (Matthews, 195Sa). Base

8-AZAGUANINE AND VIRUS INFECTIVITY 171

TABLE 2

AM~TNTS OF NUCLEIC ACID IN TYMV FROM CONTROL AND FROM S-AZAGUANINE-TREATED PLANTS

Virus from

Ana,lyses on purified preparations

( (on Pd;y%t.) Nucleic acid, %

__- Control plants 2.45 26.6 8 - Azaguanine treated 2.45 26.6

plants

- Estimates on virus in heat-clarified sap

-

0.0777 0.308 25.2 0.0427 0.163 i 26.2

analyses were made on nucleic acid prepared from TYMV isolated from crude expressed sap by precipitation with antiserum in the cold. The molar ratios of the four bases were in close agreement with those found by Markham and Smith (1951). As was to be expected with such small amounts of 8-azaguanylic acid, no significant difference in nucleic acid composition was detected between virus from control and from 8-azaguanine-treated plants. However, as free 8-azaguanylic acid has not been detected in plants treated with 8-azaguanine, it is con- cluded that the 8-azaguanylic acid found in TYMV was not a con- taminant, but was derived from the virus nucleic acid.

Ratio of virus protein to nucleoprotein. In a number of preparations isolated by a st’andard procedure from plant’s infected for 1 to 6 weeks, yields of virus varied from 0.03 to 2.0 mg/ml in the expressed sap. The Dz, for 0.1 mg/ml of purified virus varied between 0.62 and 0.81, which would correspond t’o a variation of about 2.2 to 3.0% in phos- phorus content. There was no correlation apparent between age of infection (or yield of virus) and the Ds60 for 0.1 mg,/ml of the purified preparations. The cause of this variation remains unexplained.

However, for purified virus preparations from control and from 8-aza- guanine-treated plants harvested at the same time there was no sig- nificant difference in t,he phosphorus conbent or in the optical density at 260 rnp for 0.1 mg/ml.

In one experiment, in addition to analyses of purified virus prepara- t,ions, the ratio of virus protein to nucleic acid in the plant sap was estimated by the serological procedures described earlier (Table 2).

Infectivity of Virus Containing 8-Azaguanine

Table 3 summarizes the results of comparative infectivity tests on control virus and on 8-azaguanine-containing virus. Local lesions were

172 R. E. F. MATTHEWS

Expt

-

1

2

3

-

1 I :. ,

-_

-

TABLE 3

INFECTIVITY OF TYMV CONTAINING S-AZAGUANINE

Yield of puri ied virus fro] 3-azaguaniae :reated plant

as a % of control

5.4%

22%

55%

Purified virus prepa- ration

Purified virus prepa- ration

a. Uncla rified plant

sap

b. Puri- fied virus prepa- ration

-

Method of equal- izing amount of virus material in inoculom

&A

Nucleic acid content de- termined

by sero- logical chromato- graphic method

Virus nucleic acid con- tent of sap determinec by sero- logical chromato- graphic method

Phosphorus content

-

_-

1

-

Control

8-Aza- gua- nine

Control

8-Aza- gua- nine

Control

8-Aza- gua- nine

Control

8-Aza- gua- nine

,

-

Mean nunbe] of local le- sions per half-leaf

-

I

First T

30lmt

34.:

11.1

13.i

6 .f

35.4

19.8

57.7

32.5

!i

.I

L4

i

%

COIlIll

LO.8 a

16.1 l.28**’

15.4 0

6.7t 5

1.52**

Lo.0 0.50**1

$4.9

Ratio, R'

_-

First count

io.9 0.55***

16.3

0 Log R = z[log (G + 1) - log (C + 111

, where G = number of local n lesions

on half-leaf inoculated with &azaguanine-containing virus; C = number of local lesions on half-leaf inoculated with control virus; n = number of leaves. Log R significantly different from zero (t test) as follows: * = at 0.05P; ** = at’ O.OlP; *** = at O.OOlP.

Second count

0.32***

0.54*

0.54***

0.57***

8-AZAGCANINE AND VIRUS INFECTIVITY 173

counted independently by two observers. The second observer con- sistently obtained a slightly higher local lesion count. However, the ratio 8-azaguanine/control determined on the counts made by each observer were in good agreement.

DISCUSSION

Because the amount of 8-azaguanine incorporated int’o the nucleic acid of TYMV was so small, it was not possible to estimate the ratio of Sazaguanylic acid to guanylic acid. Although base analyses could show no differences, it is probable t’hat S-azaguanine replaces a proportion of the guanine residues, as has been shown for tobacco mosaic virus (Matthews, 1954).

The infectivity of preparations of TYMV containing 8-azaguanine was less than that of t,he control virus when compared on a basis of equal virus nucleic acid. In other words, a proport’ion of the virus particles in 8-azaguanine-containing preparations are rendered in- capable of initiating infection. This suggests that in such particles the RNA is not, capable of functioning normally during virus multiplication. However, other possibilities are not ruled out. For example, it could be postulated by analogy with bacterial viruses that in the initial stages of infection by TYMV the RKA is released from t’he virus protein, and that 8-azaguanine incorporation interfered with this release of nucleic acid rather than with the reduplication of the virus.

The delay in virus production in the presence of S-azaguanine could well be explained by the format’ion of a proportion of sterile particles during virus multiplication. If a virus multiplied by a doubling process, one complete particle giving rise to two at each step, then an infect’ion would be expected to die out if more than 50% of the particles were rendered sterile during each cycle of reduplication. On the ot’her hand, if each complete virus particle gave rise to a large number of complete particles in each cycle of mult’iplication, then a much greater proportion of the particles would have to be made sterile to prevent continued virus development.

A simple mathematical formulation of this problem involves the assumptions that the proportion of particles rendered sterile at each cycle of mulbiplication is constant, and that t’he whole population of particles present is involved in each cycle. As it is very probable that neither of these conditions applies to TYMV multiplying in the presence

174 R. E. F. MATTHEWS

of &azaguanine, no inferences regarding multiplication of this virus can be drawn from the yield and infectivity data.

If, as is suggested by the experiments of Jeener (1954), the virus protein is a precursor of the complete TYMV nucleoprotein, it remains to be explained why in mature infections, where presumably little multiplication is going on, about one precursor particle is present for every two virus particles. Whatever the precise relationship between the two types of particle, 8-azaguanine treatment had no detectable effect on their relative proportions in infections where rapid virus multiplication was taking place.

Dunn and Smith (1954) showed that preparations of the bacterial virus Ttr had reduced infectivity when a proportion of the t’hymine in the DNA was replaced by 5-bromouracil or 5-iodouracil.

Comparison of their data with those obtained for TYMV might suggest t’hat 8-azaguanine was more effective than the halogenated pyrimidines in making nucleic acids into which it is incorporated non- functional. With TYMV, infectivity tests indicated that about 50% of the particles were rendered nonviable while the amount of 8-aza- guanine incorporated was probably less than 1% of the guanine. Dunn and Smith found that, when 79 % of the thymine residues were replaced by 5-bromouracil, only 70% of the T2r virus particles were nonin- fectious. As the analog was present both before and during infection, they considered that the analog was probably nearly randomly dis- tributed among the virus particles. If this were so, some virus particles containing amounts of the analog greater than 1% of the thymine would have been infectious. However, it is possible that the analog was not randomly distributed. The infective virus might have been the earlier- formed particles in which the thymine of the bacterial host was utilized for DNA synthesis (Dunn and Smith, unpublished).

REFERENCES

ALLEN, R. J. C. (1940). The estimation of phosphorus. Biochem. J. 34,858865. DUNN, D. B., and SMITH, J. D. (1954). Incorporation of halogenated pyrimidines

into the deoxyribonucleic acids of Bacterium coli and its bacteriophages. Nature 174, 305-30s.

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

JEENER, R. (1954). A preliminary study of the incorporation in growing turnip yellow mosaic virus and its related non-infective antigen of labelled amino acids. Biochim. et Biophys. Acta 13, 307-308.

S-AZAGUANINE AND VIRUS INFECTIVITY 175

KLECZKOWSKI, A. (1950). Interpreting relationships between the concentrations of plant viruses and numbers of local lesions. J. Gen. Microbial. 4, 53-69.

MARKHAM, R., and SXITH, J. D. (1951). Chromatographic studies on nucleic acids 4. The nucleic acid of turnip yellow mosaic virus, including a note on the nucleic acid of tomato bushy stunt virus. Biochem. J. 49, 401-406.

MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342.

KITTHEWS, R. E. F. (1954,). Effects of some purine analogues on tobacco mosaic virus. J. Gen. Microbial. 10, 521-532.

hI.kTTnEws, R. E. F. (1954b). Proc. 6th Intern. Congr. Microbial. 3, 1439.