DNA Viruses of Higher Plants

DNA VIRUSES

OF HIGHER PLANTS

Robert J. Shepherd

Department of Plant Pothology, University of Colifornia, Davis, California

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biological Properties of Caulimoviruses .......................... A. Diagnosis.. ...................... ....................

307

111. Physical and Chemical Properties of the Virions.. . . . . . . . . . . . . . . . .

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C. The Nucleic Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . D. Structural Proteins. . . . . . . . . . . .

B. Other Cytological Changes in Infected Cells.. . VI. Speculations on the Replication of Caulimoviruses .

311

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...........

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References. ..

I. INTRODUCTION

Until recently only RNA had been found as the genomic material of the viruses of higher plants although all other virus groups such as those of higher animals, arthropods, and bacteria are abundantly represented by both RNA and DNA types. The reason for this lopsidedness in favor of RNA for the plant viruses is an enigma that remains unanswerable at the present time. It may simply be a quirk of evolution. Nevertheless, the recent discovery th at some plant viruses do contain DNA as their genetic material has stimulated interest in these viruses. With perhaps one exception, reported recently, all the known DNA viruses of higher plants share many other attributes and hence comprise a discrete taxonomic group for which the name caulirnoviruses has been proposed (Harrison e t al., 1971). However, the potato leafroll virus, which may contain DNA (Sarkar, 1973), is altogether different in its properties and is obviously different taxonomically. The caulimovirus group contains five well-established members plus two to three other viruses as tentative members (Table I). The cauliflower mosaic virus (CaMV) and the dahlia mosaic virus (DaMV) are the best known members of the group and most of the discussion of the group herein will, 305

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ROBERT J . SHEPHERD

TABLE I PROPISRTIES OF CAULIMOVIRUSES

Virusa

Size (nm) and shape

Carnation etched ring (CERV) Cauliflower mosaic virus (CaMV) Dahlia mosaic virus (DaMV)

47,bd sphere

Mirabilis mosaic virus Strawberry vein banding virus

(%I

Melting temp. DNA ("C)

+d

-

DNA content S ~ . ~ O

-

50, spheree 208'~*

16'

254' 50, spherekJ

14.P

45-50, sphere"

254'

16-17"

40-50, sphereso

-

-

Host range

Dianthus s p . , Saponaria and SiZen@ (in Caryophyllaceae) 87s Several Cruciferae and two species of Solanaceae

Serological relationships Related to CaMV & DaMV Related to CERV & DaMVh

86" Several Cornpositae Related to and some CaMV & Amaranthaceae CERV Chemopodiaceae and Solanaceae' - Mirabilis sp. (in Unrelated to Nyctaginaceae)" CaMV or DaMVn - Fragaria sp. (in Rosaceae)p

a Cassava vein mosaic (Kitajima and Costa, 1966) and petunia vein-clearing (Lesemann and Casper, 1973) and a recently discovered virus in Scrophularia (J. E. Duffus, unpublished) may be members of the group. Hollings and Stone (1969). Rubio-Huertos el al. (1972). Pirone et al. (1961). dfijisawa et al. (1971, 1972). Itoh et al. (1969). 0 Shepherd el al. (1970). Brunt (19718, b). Hull, Shepherd, and Harvey (1976). i Kitajima et al. (1969). Brunt (1966). Brunt (1971) a, b. Gomec (1973). n Brunt and Kitajima (1973). a Kitajima et al. (1973). p Frazier (1955).

of necessity, relate t o them. Three less well-known viruses, carnation etched ring virus (CERV) mirabilis mosaic virus, and strawberry veinbanding virus, are also members. of the caulimovirus group. Cassava vein mosaic (Kitajima and Costa, 1966, 1973) is probably allied with the group and perhaps the petunia vein-clearing virus (Lesemann and Casper, 1973), although not enough is known about the latter to group it with the caulimoviruses. A virus discovered recently in Schrophularia

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307

(J. E. Duffus, personal communication) may also be a member of the group. This article is designed to survey the known properties of the caulimoviruses with the aim of pointing out their unique nature and to pr'ovide an aid for recognition and characterization of these viruses as new members of the group are encountered. 11. BIOLOGICAL PROPERTIES OF CAULIMOVIRUSES

The caulimorivuses cause mosaic-mottle types of diseases of their plant hosts that are not conspicuously different from diseases induced by an assortment of other plant viruses (Fig. 1).The diseases are economically important on a number of cultivated crops and the viruses are widely distributed throughout the temperate regions of the world. Caulimoviruses, having restricted host ranges, are confined largely t o a few closely related plants in nature, and there appears to be little if any overlap between the host ranges of the individual viruses within the group, in spite of some of the close serological affinities. The cauliflower mosaic virus (CaMV), for example, infects only members of the Cruciferae in nature and is experimentally transmissible to few plants outside this family (Tompkins, 1937). Broadbent (1957) gives a compilation of the host range, as well as a list of nonhosts, of CaMV along with good illustrations of the symptoms i t induces on infected plants. Some strains infect Nicotiana clevelandii (Hills and Campbell, 1968) or Dutura strumonium (Lung and Pirone, 1973). Similarly, dahlia mosaic virus (DaMV) is found only in Dahlia spp. in natural infections, although the virus can infect 11 other members of the Compositae and 13 species in the Solanaceae, Chenopodiaceae, and Amaranthaceae (Brunt, 1971a). Mirabilis mosaic virus has been reported to infect only M . nyctaginea and Mirabilis jalapa, although it has been inoculated to species in several other plant families (Brunt and Kitajima, 1973).

A . Diagnosis The viruses are most conveniently identified by their restricted host ranges, relatively high thermal inactivation points, aphid transmissibility, the induction of characteristic inclusion bodies, morphology, and serology. Diagnostic hosts and their reactions, and the distinguishing properties of CaMV and DaMV, have been described by Shepherd (1970) and Brunt (1971b), respectively. Carnation etched ring and mirabilis mosaic virus possess many of the same intrinsic properties of these two better known prototypes (Hollings, 1969; Fujisawa et al., 1972; Brunt and Kitajima, 1973). Sufficient information for a distinctive diagnosis of strawberry vein-banding virus is lacking except for its association with

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ROBERT J . SHEPHERD

a characteristic disease in strawberry (Fragaria chiloensis var. ananassa), and its semipersistent retention by aphids (Frazier and Sylvester, 1960) and distinctive inclusion bodies (Kitajima et al., 1973). Symptoms on infected plants generally are not sufficiently distinctive for a reliable diagnosis to be based on this alone. Nevertheless, the viruses in chronically infected plants often cause changes in leaf pigmentation associated with the major veins of the leaf (Figs. 1-5), as one might assume from some of the virus names such as cassava vein mosaic and

FIG.1-5. Symptoms of caulimoviruses on host plants. FIG.1. Cauliflower mosaic virus in turnip, Brassica campestris variety Just Right, showing chlorotic mottle. FIG.2. Strawberry vein-banding virus g m p t o m s in strawberry leaflet (photograph provided by N. W. Frazier). FIG.3. Mirabilis mosaic virus infected leaf or Mirabilis nyctaginea (photograph provided by A. A. Brunt). FIG.4. DaMV-infected Dnhlin tJarinb??Lsshowing systemic veinal chlorosis. FIG.5. CERV-infected carnation, Dianthm cnryophyllus, showing irregular necrotic spots and necrotic arcs as systemic symptoms (photograph provided by R. H. Lawson).

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309

strawberry vein-banding. CaMV, for example, in cauliflower infected for several weeks, causes a prominent interveinal chlorosis with greater retention of chlorophyll along the major veins of the leaf, to which the name “vein-banding virus” has been applied. Symptoms of DaMV in Dahlia and Verbesina encelioides are similar. In other plants, the viruses frequently induce nondescript mosaics and mottles (Figs. 1-5) that are not useful diagnostic aids, and little reliance should be placed on symptoms alone for identification of the caulimoviruses. The viruses have thermal inactivation points in the range of 75 to 8OoC, significantly higher than other aphid-borne viruses; it seems reasonable to assume th at this is a reflection of the great stability i n vitro of these viruses in comparison to the other stylet-borne viruses transmitted by aphids. The viruses are transmitted mechanically and by aphids in a styletborne manner but exhibit a somewhat anomalous behavior in the vector. This is discussed later. The caulimoviruses can frequently be diagnosed in chronically infected plants by observation of the distinctive inclusion bodies appearing in infected cells. This is most conveniently done by removing epidermal strips and staining for a few minutes in 0 5 1 % phloxine in 0.85% sodium chloride, followed by thorough rinsing with saline or water, and observation with a medium-power objective of the light microscope. The inclusions stain a deep reddish-pink and appear as well-defined spherical, elliptical, or somewhat irregularly lobed bodies in the cytoplasm, frequently near the nucleus. The nucleus may stain faintly, but generally other cell components remain colorless. Robb (1963) was the first to point out the diagnostic value of the inclusion bodies as a quick and simple means of detecting DaMV in infected plants. DaMV is considerably more difficult to transmit mechanically from Dahlia sp. to indicator hosts than CaMV is from its cruciferous hosts (Brierley, 1933; Brierley and Smith, 1950) ; hence the emphasis is placed on other diagnositc aids for the former. Lawson and Taconis (1965) reported that DaMV could be mechanically transmitted consistently, when infected Dahlia tissue was frozen in liquid nitrogen, then finely ground, and the frozen tissue powder inoculated directly to Ti. encelioides. The latter species is a useful indicator for DaMV (Brierley, 1951). SiEene nrmeria is of similar use for CERV (Hakkaart, 1968). The characteristic size of caulimovirus particles is a useful aid in the diagnosis of their diseases. Unfortunately, with the quick-dip method with extracted saps, virions are frequently difficult to find because of their low concentration in extracts. Lack of virus in extracts may be a consequence of their almost exclusive association with inclusion bodies within

310

ROBERT J. SHEPHERD

infected cells. Inclusion bodies may not break down immediately in cell homogenates to release the bulk of virus initially embedded in the matrix and this may account for the lack of free virus in ordinary extracts. For the same reason, the usual types of serological tests, useful in the identification of other viruses in saps or extracts, are of little use since the viruses are not present in sufficient amounts to give positive precipitin tests (Pirone e t al., 1961; Brunt, 1971b). As a consequence, partially purified preparations must be prepared for use with the electron microscope or for serology. Virus, when sufficiently concentrated, reacts well in gel-diffusion tests, which may be more useful than microprecipitin or tube tests (Brunt, 1971b).

B . Bioassay Convenient and reliable methods for measuring the infectivity of CaMV or DaMV are not available since dependable local lesion hosts are not known. Thus, resort must be made to systematicalIy susceptible hosts. Although D a t w a stmmonium (Lung and Pirone, 1972) and some cruciferous plants (Mamula and Milicic, 1968) have been reported as hosts giving local necrotic lesions with certain strains of CaMV, the author has had little success with these hosts. In the author’s experience most isolates of CaMV consist of a mixture of strains some of which produce discrete local chlorotic lesions on selected genotypes of B . campestris. Local chlorotic lesion-inducing strains, selected free of non-lesionproducing types, are more amenable to bioassay. Under short day conditions, V . encelioides may produce chlorotic or seminecrotic local lesions with DaMV (Brunt, 1971b). C . Serological Affinities Cauliflower mosaic virus, dahlia mosaic virus, and carnation etched ring virus (CERV) are closely related serologically (Brunt 1966, 1971a ; Hollings, 1969). Antisera to these three viruses show little differences in homologous and heterologous titers in gel precipitin tests (Brunt, 1971b). Yet the viruses have distinctly different host ranges and none infect the natural hosts of the other. Hence, in spite of their close serological relationships and other properties, they can be regarded as separate viruses. Mirabilis mosaic virus is not serologically related to CaMV, DaMV, or CERV (Brunt and Kitajima, 1973). Serological tests have not been done with strawberry vein-banding or cassava vein mosaic viruses. 111. PHYSICAL AND CHEMICAL PROPERTIES OF THE VIRIONS Members of the caulimovirus group have not been thoroughly characterized either chemically or physically, with the exception of CaMV. This lack of progress can be partially attributed t o the problem of obtaining

DNA VIRUSES OF HIGHER PLANTS

311

sufficient amounts of purified virus, since the viruses are present in the host plant in low concentrations and may not be readily released from inclusion bodies after disintegration of tissue. Moreover, the viruses show a strong tendency to aggregate and lose solubility during the course of purification. I n the case of both CaMV and DaMV, for example, i t is frequently possible to extract considerable quantities of virus from material sedimented during low-speed centrifugation. These problems have led t o the development of a new procedure for purification of the viruses (Hull, Shepherd, and Harvey, 1976). An additional obstacle to the characterization of caulimoviruses is the remarkable stability the virions exhibit to chemical degradation and the marked insolubility of the disaggregated structural proteins under ordinary physiological conditions. Development of information on the structural proteins has suffered from these disadvantages. As technical dexterity in the manipulation of these viruses develops, further studies will become feasible.

A . Morphology CaMV (Day and Venables, 1960; Pirone et al., 1960) and DaMV (Brunt, 1966) consist of spherical particles about 50 nm in diameter. Ordinary air drying of preparations on specimen grids followed by shadow casting for electron microscopic examination, is liable to give considerable flattening of virions with a significantly larger apparent diameter. Freeze drying of preparations sprayed onto grids will prevent collapse and spurious appearance of virions (Pirone et al., 1961). The particles of caulimoviruses are penetrated by potassium phosphotungstate and appear “empty” with a diameter of 50 nm and a hollow center of 20 nm diameter. I n tissue sections stained with lead and osmium salts, virions appear 45-50 nm in diameter and both cLempty”and “full” appearing particles are present. I n uranyl acetate, virions of CaMV appear 45 nm in diameter and the stain does not penetrate (Hills and Campbell, 1968) or virions may exhibit cores 25-30 nm in diameter (Kitajima et al., 1965). According t o Brunt (1971a), the virions of DaMV mounted in saturated uranyl acetate or uranyl formate are slightly swollen or disrupted, even when fixed in 3% glutaraldehyde. Virions that survive show some surface structure and an outer protein shell ca. 14-15 mm thick enclosing a hollow core ca. 20 nm in diameter. Particles remain intact but contrast poorly in ammonium molybdate. The isometric particles of carnation etched ring virus (Fujisawa et al., 1971) and mirabilis mosaic virus (Brunt and Kitajima, 1973) have an appearance and diameter similar to CaMV and DaMV. The virions of the caulimoviruses appear to be devoid of any readily discernible external substructure. Although Kitajima et al. (1965) re-

312

ROBERT J. SHEPHERD

ported that a virus in cruciferous plants in Argentina, with properties of cauliflower mosaic virus, exhibited 92 capsomeres (T = 9) in negatively stained preparations, the published electron micrographs did not convincingly reveal this structure. Data on the number of protein subunits in CaMV suggest a T = 7 shell and T = 1 core (Hull and Shepherd, 1975. The effects of slow pronase digestion on the sedimenting properties of CaMV have been studied by Tezuka and Taniguchi (1972a), who found that discrete components of lower sedimentation rate were produced before digestion had progressed to the point a t which the nucleic acid was released. From their observation they suggested that the capsid of CaMV may consist of more than one protein shell, a very tenuous suggestion based on present evidence.

B. Physical and Chemical Properties The caulimoviruses, as exemplified by cauliflower mosaic and dahlia mosaic viruses, have remarkably stable virions. As mentioned previously, they are not degraded by any of the conventional procedures employed for disruption and disaggregation of the protein capsids of viruses. Moreover, a variety of observations on the behavior of the viruses in solution suggests the exterior of the virion is very hydrophobic, a property that may account for their recalcitrant behavior to degradation. These features imply that the virions may have some unique structural properties. The author has found that CaMV is not degraded by any of the common protein-denaturing conditions [such as treatment with 5-6 M guanidine hydrochloride, emulsification with phenol, with or without cresol and/or dodecyl sulfate even a t elevated temperatures, or a single-phase phenol procedure (Diener and Schneider, 1968)] or by treatment with a wide variety of salts that denature proteins [such as lithium iodide or sodium perchlorate (Bruening, 1972; Wilcockson and Hull, 1974) 1. Proteolysis, with either pronase or fungal protease K in the presence of 0.25% dodecyl sulfate or boiling in 1% dodecyl sulfate solution is effective in destroying the virus capsid to release the DNA. For preparation of viral DNA, the latter procedure is undesirable since it causes strand separation of the double-stranded nucleic acid. Itoh et al., (1969) have documented the resistance CaMV offers to degradation a t ordinary temperatures by sodium dodecyl sulfate (SDS) , a reagent effective for disaggregation and denaturation of the structural proteins of most viruses. When CaMV is treated with 1%SDS a t 50-7OoC the virus is converted into more slowly sedimenting forms without loss of DNA, but virions revert to the original form when the SDS is removed by dialysis. The authors suggest a reversible swelling phenomenon.

DNA VIRUSES OF HIGHER PLANTS

313

CaMV contains about 17% DNA based on its phosphorus content (1.63%) and quantitative diphenylamine tests (Hull, Shepherd, and Harvey, 1976). The DNA content of DaMV has been estimated at 14.5% by diphenylamine tests (Gomec, 1973). The DNA content of the other caulimoviruses has not been determined. The sedimentation coefficients of caulimoviruses have been reported as 206 S to 250 S (Table I ) . The sedimentation coefficient of CaMV has recently been determined to be 208 in 0.1 M NaCl, 0.01 M phosphate, p H 7.2 a t infinite dilution (Hull, Shepherd, and Harvey, 1976). The s,,~, had a concentration dependence of 8 S/mg/ml up t o a concentration of 3 mg/ml. Virus banded isopycnically in cesium chloride exhibited an of 211, indicating the binding of Cs+ ion. Itoh et al. (1969) have reported an s,,~,, of 206.5 for CaMV. The partial specific volume of CaMV has also been determined as 0.704 gm/cc using a digital precision density meter (model DMA 02C, manufactured by A. Parr, K.G., Graz, Austria). of CaMV of 0.753 x m*/second has A diffusion coefficient (Da.Pc,) been determined (Hull, Shepherd, and Harvey, 1976) by a laser light scattering method (Harvey, 1973). From this value of D,,,,,, the hydrodynamic radius of the virus could be calculated to be 28.4 nm using the Stokes-Einstein equation (Einstein, 1956). Using the Svedberg equation and these values for the sedimentation and diffusion coefficients the molecular weight of the CaMV virion was calculated to be 22.8 X lo6. Hence, the DNA, comprising about 17% by weight of the particle, has a molecular weight of about 3.9 x lo6,a value somewhat lower than that estimated from the contour length of the DNA (Shepherd and Wakeman, 1971). The virions of CaMV have a buoyant density of 1.37 gm/ml in cesium chloride. The uncorrected uv extinction coefficient at 260 nm for CaMV is about 7 absorbance units/mg/ml (Shepherd, 1970). When corrected for light scattering the extinction coefficient a t 260 nm is about 4.36 absorbance units/mg/ml.

C . The Nucleic Acid CaMV has been shown to contain DNA by a variety of procedures. DaMV and CERV have also given indications for the presence of DNA. The first suggestion of DNA in CaMV was the failure to obtain the release of appreciable amounts of nucleotides after hydrolysis of weighted quantities of dried, purified virus in 0.4 hi NaOH a t 37O or 1 N HCl a t 100°C (Shepherd et al., 1968). This result indicated that nucleic acid was not RNA, which is readily hydrolyzed under these conditions. Tests of CaMV with diphenylamine were positive and further tests to correlate the presence of DNA with the distribution of infectivity and uv-absorbing material with virus centrifuged to equilibrium in cesium chloride den-

314

ROBERT J. SHEPHERD

sity gradients confirmed that the DNA was associated with virus and not some cellular organelle or other component. Subsequently, it was found that virus hydrolyzed in 70% perchloric acid yielded five times as much thymine as uracil on a molar basis. Using autoradiography Kamei et al. (1969) reported that fully expanded leaves of Brassica perviridis infected with CaMV showed a preferential uptake of tritiated thymidine centered over virus-induced inclusions, whereas healthy leaves showed no such uptake. Later reports documented the sensitivity of isolated viral nucleic acid to DNase, in contrast to the lack of effect of RNase, and a buoyant density in cesium chloride typical of DNA rather than RNA (Shepherd et al., 1970). Moreover, the multiplication of CaRilV has been found to be inhibited by actinomycin D under conditions in which turnip mosaic virus, an RNA virus, was only slightly affected (Tezuka et al., 1971). Tezuka and Taniguchi (1973) also reported inhibition of CaMV multiplication by 5-bromodeoxyuridine and 5-fluorodeoxyuridine, which inhibit DNA syntheses, and by formycin B, which also inhibits an RNA-containing virus (TMV). These data provide a convincing demonstration of DNA in CaMV. Tests for DNA with some of the other caulimoviruses, such as DaMV (Brunt, 1971a) and mirabilis mosaic virus (Brunt and Kitajima, 1973), which are more difficult to purify in any quantity, have not given positive tests for DNA. However, Dr. Belgin Gomec (unpublished results), working with the author, has shown that purified DaMV gives positive diphenylamine reactions and its nucleie acid has a bouyant density and melting behavior characteristic of double-stranded DNA. CERV is also a difficult virus to purify in any quantity. However, Fujisawa et al. (1971, 1972) have shown that the virus contains DNA by demonstrating th at it incorporates tritiated thymidine and that the isolated nucleic acid is sensitive to DNase but not to RNase. The nucleic acid can only be isolated from CaMV after the virions have been digested with pronase in the presence of SDS as mentioned earlier (Shepherd et al., 1970) (see Section 111,B ) . The properties of CaMV-DNA, which appear to be double-stranded, have been reported on by Shepherd et al. (1970), Shepherd and Wakeman (1971), and Russell et al. (1971). The viral nucleic acid shows a cooperative-type melting curve when heated in 0.15 M NaC1-0.015 M sodium citrate pH 7.0 (SSC) with a melting point (T,) of 87.2 and a hyperchromicity of 33-36% (Shepherd et al., 1970). This T, suggests a nucleotide content of about 43.5% GC (Marmur and Doty, 1965). CaMV-DNA is nonreactive to formaldehyde (1-5%) a t room temperature indicating that the amino groups are participating in hydrogen bond-

315

DNA VIRUSES O F HIGHER PLANTS

ing in a double-helical structure. When heated to temperatures near the melting point, strand separation and reactivity to formaldehyde occurred as signified by an increased hyperchromic effect (42%) and a shift in absorption to longer wavelengths. The DNA of CaMV has a buoyant density of 1.702 g/ml in cesium chloride (Shepherd et al., 1970) suggesting a GC content of about 437% (Schildkraut e t al., 1962). The same buoyant density is obtained aftermelting (lOO°C, 5 minutes) and quenching in ice water indicating that although its nucleic acid is double-stranded, it undergoes rapid renaturation, or "snap back," after melting (Shepherd et al., 1970). Although the reason for rapid renaturation is a matter for speculation, i t may be simply a reflection of the low molecular weight for the DNA. When held a t annealing temperatures (65°C) after melting the nucleic acid forms large aggregates that come out of solution as gelatinous masses unless the material is heated again a t higher temperatures and cooled rapidly. The reason for this behavior is not known but may be related to a partially single-stranded nature for the DNA. The base composition of CaMV-DNA, done using both enzymatic and acid methods for hydrolysis, showed thymine t o be a major constituent. Based on comparative R fvalues during chromatography with authentic 5'-deoxyribonucleotides, only 5'-deoxyribonucleotides were released during enzymatic hydrolysis (Shepherd et al., 1970). Both types of analyses (Table 11) with CaMV-DNA indicated a molar equivalence between dAMP and T M P and between d C M P and dCMP, thereby supporting the other criteria for double-strandedness (Table 11). Nucleotide analyses TABLE I1 NUCLEOTIDE RATIOS I N C.4ULIFLOWER MOSAICVIRUS DNA"'b Type of hydrolysis of nurleic acid

dAMP

dGMP

dCMP

TMP

(%I

Enzymaticc Acidd Average

28.3 29.9 29.1

20.8 20.1 20.5

22.1 20.7 21.4

28.8 29.3 29.1

43 41 42

G plus C indicated

Moles per 100 moles of nucleotides

nShepherd et al. (1970). = 0.98, &amino bases/&keto bases = 1.02. Values are the average of three experiments with pancreatic deoxyribonuclease and venom phosphodiesterase. Values are the average of two experiments after hydrolysis with either formic or perchloric acid.

* pulpy

316

ROBERT J . SHEPHERD

showed a GC content of 4 1 4 3 % , similar to that indicated by T , and buoyant density data. No evidence for unusual bases was obtained in nucleotide analyses. Neither 5-methylcytosine, common in leaf DNA of higher plants, or 5-hydroxymethylcytosine, preserved by hydrolysis with 90% formic acid (Wyatt and Cohen, 1953), was revealed by either type of hydrolysis. In this respect the nucleic acid resembles chloroplastic DNA, which lacks 5-methylcytosine (Temari and Wildman, 1966). A nearest-neighbor frequency analysis showed that CaMV-DNA has a chemical structure showing close similarities to that of host cauliflower DNA (Russell et al., 1971). Electron microscope studies of CaMV-DNA (Shepherd and Wakeman, 1971 ; Russell et aE., 1971) showed two types of molecules (Fig. 6 ) , circular and linear, each with the same contour length of 2.31 m (Shepherd and Wakeman, 1971) or 2.47 pm (Russell e t al., 1971); these lengths correspond to molecular weights of 4.4 x lo6 and 4.7 X lo6, respectively. No supercoiled molecules were observed, which is consistent with the fact that CaMV-DNA forms only one band in cesium chloride in the presence of ethidium bromide (Shepherd et al., 1970). Thus it appears th a t extracted CaMV-DNA exists as nicked-circular and linear forms. Freshly isolated CaMV-DNA sediments as two components of 18 S and 20 S (Russell et al., 1971). Hull and Shepherd (unpublished observations) measured the sedimentation coefficients to be 17.1 S and 19.8 S. The conversion of the faster to the slower compound by mild DNase digestion indicated that the faster component was the nicked-circular form and the slower component the linear form. From the sedimentation coefficient of the linear form, the molecular weight can be estimated to be about 4 x lo6.The two forms can be separated by electrophoresis on polyacrylamide gels and from such separations and from separation on sucrose density gradients it appears that only the nicked-circular form is infectious (Hull and Shepherd, 1975). There is no evidence from either electron microscopy or from sedimentation analysis of forms other than the two mentioned (i.e., no covalently closed circles). Thus it would seem likely that the nicked-circular form is the natural form and this is further degraded to give the linear molecules. The possibility of the DNA being linear and circularizing after liberation from the virion cannot be ruled out (Shepherd and Wakeman, 1971). However, the infectivity data tend to support the first possibility. CaMV-DNA that has been treated with alkali sediments as a somewhat heterogeneous peak a t about %10 S (Hull and Shepherd, unpublished). This indicates a single-stranded molecular weight of 4-5 x lo5 which is considerably less than the natural single strand. Since treatment with other

3a

20

d I Ei a

# I 1

A

B

C

D

I

15

10

5

C

DNA LENGTH ( p )

FIG.6. Top: Electron micrograph of DNA from CaMV showing circular and linear molecules. Bottom : Histograms showing contour length distribution of four preparations of CaMV DNA. (From Shepherd and Wakeman, 1971.)

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ROBERT J. SHEPHERD

denaturants, e.g., heat and formaldehyde, does not reveal hidden breaks it seems as though CaMV-DNA is made up of small pieces linked by alkali labile regions. This and other evidence (Hull and Shepherd, unpublished) indicate that the nucleic acid of CaMV has appreciable quantities of RNA covalently bonded to the DNA.

D. Structural Proteins Recent investigations with the structural proteins of CaMV reveal several distinctive polypeptides in the virion. It has not been possible to obtain soluble solutions of these in the absence of dodecyl sulfate. Hence, it has not been practical to separate the proteins and study their properties individually. Tezuka and Taniguchi (1972b) reported two proteins with molecular weights of 33,000 and 68,000 from dodecyl sulfate degraded CaMV. In addition, some higher molecular weight components were detected in small amounts but were believed to be aggregation products of the major polypeptide constituents since they were not detected when the virus was degraded with both dodecyl sulfate and 8 M urea. I n a similar investigation with CaMV by Kelley et al. (1974) two major polypeptides with molecular weights of 32,000 and 67,000 were obtained with an approximate molar ratio of 4 to 1 for the smaller to larger proteins. In addition, very small amounts of four other proteins with molecular weights of about 27,000, 40,000, 92,000, and 100,000 were separated. The two largest proteins gave positive reaction with Shiff’s reagent, suggesting they were glycoproteins. More recently with CaMV Brunt et al. (1975) reported molecular weights of 42,000 and 67,000 for the major structural proteins and approximately a 5 to 1 molar ratio for the smaller to larger polypeptides. Although minor amounts of several higher molecular weight components were separated, none of these gave positive tests for glycoproteins. Both Kelley et al. (1974) and Brunt et al. (1975) observed an apparent conversion of some of the higher molecular weight proteins to lower molecular weight components, indicating that some of the latter were degradation products. I n yet another investigation on the structural proteins of CaMV (Hull and Shepherd, 1975) four polypeptides were separated consistently and two others occasionally. One of the latter was shown to be a degradation product of one of the major polypeptides. The major components, with molecular weights of 37,000 and 64,000, were present in a molar ratio of about 5 to 1 and made up more than 90% of the viral protein. The two highest molecular weight proteins (96,000 and 88,000) gave feint but indecisive staining for a glycoprotein with the periodate-Shiff proce-

319

DNA VIRUSES OF HIGHER PLANTS

dure. With 32Plabeling procedures, no evidence was obtained for phosphorylation of the polypeptides or for the presence of nucleic acid in the gel bands. Brunt and Kitajima (1973), working with mirabilis mosaic virus, a much more difficult virus to handle, found but a single protein of 32,000 daltons after SDS degradation. The various polypeptide components and their molar ratios reported by various investigators for the structural proteins of CaMV are given in Table 111. From the values for the major components and the molecular weight equivalent for the protein of cauliflower mosaic virions given above, one can calculate that each virion has about 418 copies of the 37,000 dalton protein and about 55 copies of the 64,000 dalton protein, suggesting a T = 7 structure with a T = 1 core (Hull and Shepherd, 1975). The various investigations on the structural protein of CaMV show that a t least four proteins are present and perhaps minor amounts of others. Some of the latter might be host proteins, such as of the minor proteins in polyoma virions, which have been shown to be host derived (Frearson and Crawford, 1972). TABLE I11 MOLECULAR WEIGHTS A N D MOLAR RATIOSOF CAULIFLOWER MOSAICVIRUS STRUCTURAL PROTEINS Kelley et al. (1974) Protein designation

(XW)

Molar ratio

1 2 3 4 5 6

106 92 67 40 32 27

0.01 0.18 0.07 0.69 0.06

0

Mol . wt.

Value not given.

0.02

Hull and Shepherd (1975)

Brunt et al. (1975) Protein designation

( x 106)

1 2 3a 3b 4 5 6 7a 7b 8 9 10

91 88-85 70 65 62 55 48 42 39 33 28 15

Mol. wt.

Protein Mol. Molar Molar desig- wt. ratio nation ( ~ 1 0 6 ) ratio

0.2 0.12 0.62 0.06

3 4

71 64

0.15

6

37} 33

0.77

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ROBERT J . SHEPHERD

I n addition to investigating the various species of polypeptides in virions of CaMV, Brunt et al. (1975) did amino acid analyses of whole virus. These analyses revealed that CaMV has an unusually high content of basic amino acids. For example, lysine and arginine together made up about 23% by weight of the protein. Although it is not known yet how these amino acids are distributed among the various proteins, it is obvious that some of the proteins are very basic and are probably located internally in the virion in association with the nucleic acid. IV. TRANSMISSIBILITY The caulimoviruses are transmitted by aphids in nature and this appears t o be their sole means of dispersal, with the exception of some spread with vegetatively propagated host material. The latter undoubtedly applies in the case of DaMV, which is disseminated widely by infected dahlia tubers, and with strawberry vein-banding virus in strawberry during its clonal propagation. Caulimoviruses are transmissible mechanically but some hosts are not readily infected by mechanical means. Dahlia pinnata, for example, is not readily infected by DaMV by mechanical inoculation but is readily infected by aphids (Brunt, 1971b). A low percentage of M . jalapa seedlings are infectible by mechanical inoculation with mirabilis mosaic virus (Brunt and Kitajima, 1973). DaMV is not readily transmitted from Dahlia species to the indicator V . encelioides except by special techniques (Lawson and Taconis, 1965). None of the viruses are seed-borne in their hosts.

A . Insect Transmission Aphids are the vectors of caulimoviruses and relatively little specificity is exhibited between virus transmission and aphid species. At least 27 species of aphid, for example, are reported to transmit CaMV (Kennedy et al., 1962), and 13 species are reported to transmit DaMV (Heinze, 1951, 1952 ; Brierley and Smith, 1950). Mirabilis mosaic and strawberry vein-banding viruses are also readily transmissible by aphids. Insect transmission characteristics of the caulimoviruses, as exemplified by CaMV and DaMV, indicate the viruses are probably nonpersistent, or stylet-borne by their aphid vectors. However, such inconsistent results have been obtained with CaRlV that one cannot be too unequivocal in this matter. I n early studies the viruses exhibited features typical of conventional stylet-borne viruses. The viruses, for example, were reported to be acquired rapidly during feeding periods of 1 to 10 minutes, to be immedi-

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ately transmissible to healthy plants, and to be retained for less than 3 hours (Brierley and Smith, 1950; Caldwell and Prentice, 1942; Severin and Thompkins, 1948). Similarly, Day and Irzykiewicz (1954) showed that CaMV could be acquired and inoculated by either Myzus persicae or Brevicoryne brassicae in a total elapsed transmission time of less than 2 minutes. Van Hoof (1954) reported, however, that cauliflower mosaic virus transmission by Myzus persicae and Brevicoryne brassicae exhibited features of both nonpersistent (stylet-borne) and persistent (circulative) viruses (as defined by Watson, 1946). Virus inoculativity was retained by insects through four to five consecutive feeding periods of 5 minutes each, in contrast to the usual nonpersistent, behavior in which inoculativity is retained only through the initial feeding act following acquisition. Moreover, neither aphid showed a response t o preacquisition fasting as do conventional nonpersistent or stylet-borne viruses. Transmission efficiency was the same with long acquisition feeding periods as with brief acquisition feeding periods with or without preliminary fasting. Hamlyn (1955) in a similar investigation with these two aphid species and CaMV found some response to preacquisition fasting of 15 to 30 minutes but the response was less than that obtained with typical nonpersistent viruses. She confirmed van Hoof's results on persistence of inoculativity through several consecutive transfers on healthy plants. The virus was retained by feeding aphids for more than 3 hours, and neither preacquisition fasting nor duration of the infection feeding period markedly influenced transmission in sharp distinction to the usual nonpersistent virus. From those preliminary accounts i t became obvious that cauliflower mosaic might be an unusual case of stylet transmission, a feature confirmed by subsequent investigations. Chalfant and Chapman (1962) made an elaborate study of the aphid transmission characteristics of turnip mosaic virus and CaMV with the former serving as a typical example of nonpersistent or stylet transmission. Both M . persicae and B . brassicae were used as vectors and remarkable differences in transmission were found between these two aphid species as well as the two viruses. As expected, both insects transmitted turnip mosaic virus with features characteristic of a conventional styletborne virus, viz., a marked response to preacquisition fasting and short acquisition feeding periods plus loss of inoculativity during initial feeding activity. In contrast to the observations of van Hoof and Hamlyn, however, M . persicae transmitted CaMV in a typical nonpersistent manner, with less transmission after longer acquisition feeds and short retention regardless of the duration of acquisition feeding. Conversely, B. brassicae transmitted CaMV efficiently after long periods on virus source plants and exhibited two acquisition intervals for most efficient transmission-a

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short period of about 5 minutes and a long period of 5 to 8 hours-to produce a bimodal efficiency curve when percent transmission was plotted against acquisition feeding period (Chalfant and Chapman, 1962). The bimodal curve suggests two types of transmission of the virus by B. brassicae, occurring simultaneously : the first period represents a nonpersistent or stylet-borne phase and the second a persistent or circulative type of transmission. I n comparative tests for retention in insects starved or fed after virus acquisition, B. brassicae retained inoculativity 10-20 hours when feeding, regardless of the duration of acquisition feeding, but exhibited prolonged retention during postacquisition starvation if insects had been given a long acquisition feeding period. After a 5-minute acquisition feeding period inoculativity was retained for more than 16 hours during post-acquisition starvation, or more than 36 hours if given an initial 24-hour acquisition feed. Although the duration of acquisition feeding obviously had some effect on retention, the significance of this is not obvious. Although it can be interpreted as suggestive of persistent or internal transmission, this is only tentative since greater stability of virus, or more likely of some accessory factor required for transmission (see Section IV, B) , could explain this behavior. Heinze (1959), for example, observed that aphids a t temperatures of about O°C retained inoculativity with turnip mosaic virus for up to 6 days. I n a n effort to substantiate that CaMV is retained internally by B . brassicae, Chalfant and Chapman (1962) allowed viruliferous aphids to probe into solutions of formalin with the aim of using the aldehyde as a selective inactivator for stylet-borne virus. I n tests with insects given various acquisition feeding periods, probing into 1.25% formalin rendered aphids nonviruliferous after short (2 minutes) acquisition feeding periods but was ineffectual after long (1 to 2 day) acquisition. I n another type of test in which the distal 30 pm of bared stylets were dipped directly into 0.25% formalin, a similar association between acquisition feeding period and inactivation was found. Hence these observations implied an internal type of retention for CaMV in B. brassicae. I n somewhat similar trials with CaMV and B. brassicae, Orlob and Bradley (1961) reported that no transmission occurred if the bared tips of aphid stylets were dipped into 0.25 or 1% formalin for 30 seconds after the aphids had fed overnight on infected plants, whereas 40% of the individuals transmitted if stylet tips were inserted into water. They also found that ultraviolet irradiation of the distal 15 pm of the stylet tips rendered aphids nonviruliferous with CaMV. The experiments of Day and Venables (1961) have a bearing on the long retention of CaMV by B. brassicae. They injected purified CaMV into the hemolymph of M . persicae and B. brassicae without inducing

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inoculativity when the insects were placed for a 3-day test period on indicator plants. Similarly unsuccessful was the injection of hemolymph taken from B brassicae bred on virus-infected plants into the hemolymph of nonviruliferous B. brassicae. These results do not support internal transmission of CaMV by aphids. Moreover, aphid vectors can readily acquire inoculativity of internally borne viruses following uptake of virus through a membrane, but CaMV cannot be acquired in this way (Pirone and Megahed, 1966), except under special conditions (Lung and Pirone, 1974) . I n other relevant experiments Day and Venables (1961) found that B. brassicae could retain inoculativity of cauliflower mosaic virus for 3 days but that retention never followed a molt during which the stylets are shed along with the cast off exoskeleton. This confirmed a previous experiment by Day and Irzykiewicx (1954) for the loss of virus during ecdysis of M . persicae. From these various investigations one is led to believe that transmission is altogether stylet associated in nature, and that the extended retention of inoculativity, including the lack of influence of preacquisition starvation and postacquisition feeding activity on transmission efficiency, are but anomalous manifestations of stylet-borne virus transmission rather than an unorthodox relationship between virus and vector. This notion has been reinforced by the recent reports of Lung and Pirone (1973, 1974) on the participation of an accessory factor for vector transmission of CaMV. Aphid transmission characteristics for caulimoviruses other than CaMV have not been thoroughly investigated. DaMV is known to be retained by aphid vectors for about 3 hours (Brierley and Smith, 1950). Strawberry vein-banding has been found to be retained by aphids in a semipersistent manner with a half-life of about 10 hours (Frazier and Sylvester, 1960) but, like CaMV, it is not retained over a molt of an aphid vector (Frazier, 1966). The efficiency of aphid transmission of strawberry vein-banding increases with increasing duration of the acquisition feeding period and inoculativity is not lost in the first feeding activity after acquisition (Frazier, 1955). Hence this caulimovirus is very similar t o CaMV in its aphid transmission relationships. The reason for the unusual behavior of caulimoviruses in their aphid vectors is not known. Day and Venables (1961) have suggested it may be related t o the unusual stability of these viruses in comparison with other stylet-borne viruses. A more reasonable inference is that the accessory factor required for retention is more stable than the accessory factors produced by some of the other stylet-borne viruses. Like stylet-borne viruses in general, there seems to be little overt specificity between vector and virus. As mentioned previously, many aphid

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species will transmit CaMV or DaMV (Kennedy et al., 1962; Heinze, 1951, 1952; Brierley and Smith, 1950).

B. Accessory Factor f o r Aphid Transmission The most interesting recent development on the insect transmission of the caulimoviruses is the reports by Lung and Pirone (1973, 1974) on the need for an additional virus-specified factor for aphid transmission. Several years ago, Pirone and Megahed (1966) found that CaMV was not transmitted by aphids allowed to probe into purified virus solutions through a membrane, although certain other stylet-borne viruses were transmitted under these conditions. The reasons for this lack of transmission were not known until recently, when some revealing experiments were done with nontransmissible isolates of CaMV. Lung and Pirone (1973, 1974) found that some nontransmissible isolates of CaMV could be transmitted by aphids if these were provided to aphids in plants simultaneously infected with aphid-transmissible isolates of CaMV. In other experiments, aphids could acquire and transmit the normally nontransmissible isolates if allowed to probe previously into plants infected with a transmissible isolate. These results suggested that aphids obtained some additional factor from infected plants th at was required for aphid transmission and that aphid nontransmissible isolates lacked the ability to induce this factor in infected plants. This factor has not been isolated from infected plants nor its properties defined. These observations imply th at a much more intimate biological relationship occurs between aphid and virus than previously foreseen, and that the virus specifies a gene product, probably a protein, that determines aphid acquisition and dissemination of the virus in nature. From evidence for similar accessory factors that has been found for the styletborne aphid-transmitted potyviruses (Kassanis and Govier, 1971), one can surmise that both virus groups have probably evolved a mechanism t o ensure their dispersal by aphids in nature. Knowledge of the accessory transmission factors of potyviruses and caulimoviruses might greatly improve our understanding of the transmission of these stylet-borne viruses in nature. Since viruses transmitted in this fashion incite the most numerous and damaging group of virus diseases affecting cultivated crops, emphasis should be placed on seeking out these factors and documenting their specific function in transmission.

V. CYTOPATHOLOGICAL EFFECTS OF CAULIMOVIRUSES A distinctive feature of the diseases incited by caulimoviruses is the presence of a characteristic type of inclusion body in infected cells. These inclusion bodies are unlike any produced by other plant viruses and a

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variety of observations suggest they play an important role in replication of the viruses. I n addition, a variety of other cytological effects occur, especially aberrations of the cell walls.

A . Virus-Induced Inclusion Bodies With the light microscope the inclusion bodies of CaMV of DaMV appear as compact, highly refractive masses in the cytoplasm of infected cells (Rubio-Huertos, 1950; Robb, 1963). They may be elliptical, ovoid shaped, or irregularly lobed. Some investigators have applied the term “spherule” after Littau and Black (1952) for similar appearing inclusions induced by wound tumor virus, or “X-bodies” after Goldstein (1927), who was the first to describe inclusion bodies in dahlia infected with a mosaic virus. Usually one, but perhaps several of the inclusions develop per epidermal cell 2 4 weeks after infection. With the light microscope they have a smooth, well-defined surface and are very refringent. They may appear very finely vacuolated or more coarsely so in the case of DaMV (Robb, 1963). In general, DaMV induces spherical, more uniform inclusions than CaMV, but the size and shape of the inclusions of either virus may vary with different host plants, virus strains, or the duration of infection (Mamula and Milicic, 1968) . Inclusion bodies are particularly conspicuous in stripped epidermis stained with 0.5-1% phloxine (Fujisawa et al., 1967), trypan blue in water or 1% NaCl as described by McWhorter (1941), but can be seen well with interference contrast optics without staining (Martelli and Castellano, 1971). Inclusions develop throughout various types of leaf cells, being common in epidermis, palisade and spongy parenchyma, and to a lesser extent in young tracheary and phloem companion cells. Inclusion bodies are less well-developed or conspicuous in Dianthus spp. infected with CERV (Rubio-Huertos et al., 1972), than in the case of CaMV and DaMV in their hosts. Inclusions vary in size with the duration of infection. I n general, they increase progressively in size during the course of infection (RubioHuertos, 1956; Mamula and Milieic, 1968). Those of DaMV are generally 0.2-10 pm in diameter (Robb, 1964; Kitajima et al., 1969) whereas those of CaMV may reach 20 pm in length (Mamula and Milicic, 1968). I n contrast, some strains of CaMV brought to the attention of the author by Drs. M. C. Phatak and N. Paludan induce very small, inconspicuous inclusions, about the size of mitochondria, which are not detectable with the light microscope. The inclusions of caulimoviruses occur predominantly, if not solely, in the cytoplasm of infected cells, although they are frequently found near the nucleus or dictyosomes (Figs. 7 and 8). Robb (1964) claimed

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FIG.7. A typical vacuolated conglomerative inclusion in a leaf cell of Zinnia elegans infected with DaMV. Several small protoinclusions, consisting entirely of matrix material, and some dictyosomes can be seen in the cytoplasm near the large inclusion. The inset shows fine structures of the matrix and virions (arrows). v, virions; cw, cell wall; d, dictyosomes. (From Kitajima et nl., 1969.)

to find inclusions of DaMV in the nuclei of infected D. variabilis and suggested that perhaps they arose there and were subsequently extruded into the cytoplasm. Similarly, Fujisawa et al., (1967) observed and illustrated what were reputed to be CaMV inclusions in the nucleus of Raphanus sativus. Allegations for the occurrence of intranuclear inclusions were based on light microscopic observations and have never been substantiated in numerous investigations with the electron microscope.

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One wonders if prominent, unusually large, nucleoli that stain similarly t o virus inclusions may not have been mistaken for the latter. Nucleolar material may also account for the occasional patches of electron-dense material observed by Conti et al. (1972) in CaMV-infected cells. Virions of CERV have been observed in electron microscopic observations of infected Dianthus barbntus to occur in nuclei in the case of both early and chronic infections, but no inclusion bodies have been found in nuclei of infected plants (Rubio-Huertos et al., 1972; Lawson and Hearon, 1973). I n studies with the electron microscope it is clear that caulimovirus inclusions never possess an external membrane (Fig. 7 ) . In early observations with the light microscope the inclusions were mistakenly believed to have an external membrane or nonstaining pellicle (Rubio-Huertos, 1956; Robb, 1963). The observation of ultrathiri sections with the electron microscope has shown a well-defined unique structure for caulimovirus inclusion bodies. The bulk of a typical inclusion consist of an electron-dense, granular matrix in which virions are embedded (Fujisawa et al., 1967). Throughout the matrix, in an apparent random distribution, are transparent, vacuolelike areas of roughly circular outline 0.1-1 pm in diameter (Kitajima et al., 1969) (Fig. 7 ) . These vacuoles may compose a reticulate system of lacunae that radiate throughout the inclusion bodies (Robb, 1964). Virions occur within and clustered around these vacuolelike areas as well as embedded within the matrix. There is some tendency for virus particles t o crowd near or in vacuolar spaces (Fujisawa et al., 1967). I n lacunae without virions a very sparse network of fine fibrils can be seen with high resolution. A vacuolated, conglomerative type of inclusion body (Fig. 7) is unique to the caulimoviruses and provides a reliable characteristic for distinguishing these viruses for other aphid-borne viruses. A striking observation that has emerged from studies on the intracellular distribution of the caulimoviruses in their hosts is the almost complete association of virus with inclusion bodies and the notable lack of virus elsewhere in infected cells. I n an early study of the intracellular distribution of CaMV carried out by Day and Venables (1961) with a fluorescent antibody technique, virus antigen was confined to discrete foci in the cytoplasm of infected leaf cells, except for a few phloem cells in which the whole cytoplasm was stained. Neither nuclei nor chloroplasts ever gave a positive test for viral antigen. Fujisawa et al. (1967) in an electron microscope study of CaMV-infected plants remarked upon the almost total restriction of virus to inclusion bodies within the cell and stated that this appeared to be a characteristic of CaMV infection. This has

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FIG.8 . A cell of DaMV-infected zinnia with several small inclusions, including some with virions, showing peripheral ribosomes (r) and the close association of inclusions with dictyosomes (d) ; mv, microvesicles. (From Kitajima et al., 1969). FIG.9. A preparation of CaMV inclusions isolated by the author.

been amply confirmed by subsequent investigators for CaMV and other caulimoviruses. Only occasionally have solitary or a few scattered virions of CaMV or DaMV been found within cells t h a t are not associated with the granular matrix material of the inclusion (Rubio-Huertos et al., 1968a; Brunt, 1969; Kitajima et al., 1969; Martelli and Castellano, 1971; Lung

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and Pirone, 1973). However, individual particles of CaMV, DaMV, and CERV without matrix material do occur frequently in plasmodesmata (Kitajima and Lauritis, 1969; Conti et al., 1972; Kitajima et al., 1969) (Figs. 9-13). A restricted distribution may not be true for CERV t o the same extent as CaMV or DaMV, as virions of CERV have been found individually in cytoplasm, and perhaps nuclei, of D. barbatus (Rubio-Huertos et al., 1972) and Saponaria vaccaria (Lawson and Hearon, 1973). Virions of caulimoviruses have never been found in other cell organelles, such as chloroplasts or mitochondria, or associated with endoplasmic reticulum. The cytopathological effects of DaMV, CERV, mirabilis mosaic virus, and strawberry vein-banding virus are very similar, with minor exceptions, to those of CaMV-infected cells (Kitajima et al., 1969, 1973; Fujisawa et al., 1971; Brunt and Kitajima, 1973; Lawson and Hearon, 1973). All the viruses induce dense, cytoplasmic inclusions with virions embedded in the matrix of the body and cause conspicuous changes in cell walls and plasmodesmata (see Section V, B ) . The number of virions per inclusion body varies enormously and is not necessarily associated with the size of the body. Some inclusions are packed with virions, while others have only a few scattered particles throughout the matrix. An unusually high density of virions in DaMVinduced inclusions has been exhibited by some isolates (Petzold, 1968). Inclusions induced by CERV in D. barbatus, D. curyophyllus, or S. vaccariu differ from those of CaMV or DaMV in having less granular material and proportionately more virions (Rubio-Huertos et al., 1968a,b; Fujisawa et al., 1971; Lawson and Hearon, 1973). In fact, in some cells, virions of CERV can be found with little, if any, of the granular matrix of the typical inclusion; in other cases the dense areas of the inclusion are composed mainly of closely packed virions (Rubio-Huertos et al., 1972). Cytoplasmic inclusions stained with uranyl or lead salts exhibit two types of virions; one has a dense uniformly stained particle, the other appears as an outer heavily stained shell surrounding an empty inner core (Fujisawa et al., 1967). With the latter the particle shows an outer stained layer about 10 nm thick around a central hollow core ca. 20 nm in diameter. Since time of exposure and p H affect the penetration of electron stains (Davison and Francki, 1969) it may be hazardous to conclude there are two types of particles within inclusion bodies. However, Conti et al. (1972) reported that after DNase digestion all virions had an “empty” appearance. The virions in situ measure 42-48 nm in diameter for CaMV, DaMV, or CERV (Martelli and Castellano, 1971; Kitajima e t al., 1969; Rubio-Huertos et al., 1972). Virions within inclusions appear

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to have a diameter somewhat smaller than virus in vitro and may have a more compact structure. This has been noted for various other viruses in situ. Virions never occur in packed crystalline or hexagonal arrays within cells even in inclusions with numerous virions. The matrix of the inclusion body appears to consist of an irregular aggregated mass of finely graunular, amorphous material, which appear to be largely protein based on its susceptibility to proteolytic enzymes (Martelli and Castellano, 1971; Conti et al., 1972) and staining properties (Rubio-Huertos, 1956; Robb, 1964; Kitajima et al., 1969). It makes up the bulk of inclusion of CaMV and DaMV and seems to comprise a self-coherent mass. Substructure consisting of parallel electron-dense fibers ca. 70 A thick has been visualized a t high resolution of the matrix material (Kitajima et al., 1969) (Fig. 7, insert). Very fine fibrils have been noted in the cell nucleus and neighboring areas of the cytoplasm close to virus inclusions (Martelli and Castellano, 1971). I n spite of the abundance of virions in inclusion bodies they give a negative Feulgens reaction for DNA (Rubio-Huertos, 1956; Kitajima et aE., 1969; Martelli and Castellano, 1971). Conversely, inclusion bodies may be rich in RNA since a marked difference occurs in their staining properties before and after treatment with RNase (Robb, 1964; Kitajima et al., 1969) and, in general, inclusions stain remarkably like nucleoli, which are known to be rich in RNA (Martelli and Castellano, 1971). Incipient inclusion bodies appear as minute patches of matrix material in the cytoplasm of infected cells. Several or many of these electron-dense areas may occur in the cytoplasm of a single cell, with numerous ribosomes clustered around the periphery of each (Kitajima et al., 1969; Martelli and Castellano, 1971; Lawson and Hearon, 1973) (Fig. 8 ) . These observations, plus evidence for the protein nature of the matrix, indicate t ha t incipient inclusions are sites of intense protein synthesis. As these centers enlarge, virions appear within the interior of the body embedded in the matrix material as if these are sites for virus synthesis and assembly. Since most chronically infected cells contain a single or rarely a very few large inclusions, surrounded by several small masses of matrix material, one is led to believe that the large bodies are formed by progressive aggregation and coalescence of the smaller units (Kitajima et al., 1969; Lawson and Hearon, 1973). One can surmise that growth is primarily by accretion of matrix material synthesized in the surrounding cytoplasm. The apparent cohesion of the matrix material suggests that it possesses a strong tendency for self-aggregation. The inclusion bodies are quite stable to manipulation as noted by Rubio-Huertos (1956) in his early observations of those induced by CaMV. He found that intact bodies could be dissected out of cells without previous fixation and that they were not solubilized by water, alcohol,

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chloroform, xylol, or low pH, but swelled in alkali. Some strains of CaMV have more stable inclusions than others and procedures have been developen for their isolation (Shepherd, Purcifull, and Howorth, unpublished). An electron micrograph of isolated inclusions is shown in Fig. 9.

B. Other Cytological Changes in Infected Cells A variety of cytological disturbances, in addition to inclusion bodies, have been observed in caulimovirus infected cells. Rubio-Hertos et al. (1968b) reported that although chloroplasts of Brassica oleraceae infected with CaMV were normal in size and shape, their matrices were denser than normal and their grana and lamellae were not clearly distinguishable. I n addition, mitochondria were smaller and showed a loss of cristae. I n Brassica chinensis with the same virus, Conti et al. (1972) found no change in chloroplasts but observed nuclei t o have enlarged nucleoli that contained masses of deeply staining granular material appressed to the inner nuclear membrane. Moreover, mitochondria of infected cells were smaller with an electron-transparent matrix and the cell wall formed prominent protrusions. Dilated endoplasmic reticulum and small vesicles are a cytopathological feature of CERV-infected tissues (Lawson and Hearon, 1973). Inclusions are believed to develop in association with dictyosomes as reported by several investigators. Both Petzold (1968) and Kitajima et al. (1969) have observed the presence of numerous dictyosomes or stacked Golgi cisternae adjacent to DaMV inclusions in infected cells. Kitajima et al. (1969) observed that dictyosomes (Golgi bodies) were more frequently found in association with smaller inclusions and that their number decreased as the size of the inclusion increased as if there were some relationship in their development. Others have reported hypertrophy of dictyosomes in cells infected with CaMV (Bassi et al., 1974). The latter investigators also found numerous microvesicles, 100-200 nm in diameter, in association with the dictyosomes (Figs. 7 and 8 ) , this trait being similar to CERV-infected Saponaria barbatus (Lawson and Hearon, 1973) or B. barbatus (Rubio-Huertos et al., 1972). The inclusion bodies of mirabilis mosaic and strawberry vein-banding viruses are often spatially associated with Golgi bodies in the cell (Brunt and Kitajima, 1973; Kitajima et al., 1973; Rubio-Huertos et al., 1972). Conti et al. (1972) have pointed out that cell wall protrusions are associated with convoluted membranes and vesicles in CaMV-infected plants. It is plausible that prominent dictyosomes and microvesicles are reflections of virusinduced qbnormalities in cell walls and plasmodesmata since the function of dictyosomes is to produce cell wall polysaccharides and glycoproteins (Gardiner and Chrispeels, 1975). Kitajima and Lauritis (1969) have found that DaMV causes a very

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characteristic morphological transformation of some plasmodesmata in infected cells of Z. elegans (Figs. 10-14). Although the majority of plasmodesmata of infected cells had the same basic structure and dimensions as those in healthy tissue, about 1%) had undergone enlargement with disappearance of the desmotubule ; some exhibited the development of a large papilla around the opening of the plasmodesmata where it extends into the cytoplasm (Figs. 10 and 1 1 ) . The lumen of modified plasmodesmata was 60-80 nm in diameter in contrast to 25-35 nm for the normal type. Desmotubules were not observed within the lumen of modified plasmodesmata, and the endoplasmic reticulum normally associated with plasmodesmatal openings was also lacking. Frequently, virions and the finely granular, electron-dense material characteristic of the inclusion body matrix were observed within the lumens of transformed plasmodesmata (Figs. 10-14). No transformed plasmodesmata without virions were observed, suggesting that modification was temporary during the transport of virions to neighboring cells. This seems unlikely though. Somewhat similar abnormal plasmodesmata containing virions and various sorts of cell wall protrusions, some associated with vesicles and convoluted membranes, have been reported for CaMV-infected Brassica (Conti e t al., 1972) and CERV-infected Saponaria (Lawson and Hearon, 1973). I n a recent autoradiographic study of CaPvlV-infected cells Bassi et al. (1974) administered glucose-3H and showed that the radioactive label was fixed preferentially in the large cell wall protrusions and associated vesicles. No such fixation in cell wall associated structures occurred in healthy tissue with g l u c o ~ e - ~ Hthe , immediate precursor of cell wall polysaccharides. Hence, infection seems to stimulate the host cell to synthesize additional cell wall materials to construct the protrusions and their associated plasmodesmata. These cytological changes make one wonder if virus-specified mechanisms are involved in the cell-to-cell movement of these viruses.

REPLICATION OF CAULIMOVIRUSES Little relevant information is available on the replication of caulimoviruses, but some educated guesses can be made about the events that VI. SPECULATIONS ON

THE

FIGS.10-14. Longitudinal sections of “transformed” plasmodesmata containing virus particles in leaf cells of DaMV-infected zinnia. Figures 10 and 11 show cell wall protrusions extending into the cytoplasm surrounding the ends of the enlarged plasmodesmata. Figure 13 shows a partially transformed plasmodesmata. The top half is about normal in size. Figure 14 shows a transverse section of a transformed plasmodesmata with enclosed virion. A thin d m s e laycr (arrow) is distinguishable below the plasmalemma. cm, cell membrane ; er, endoplasmic reticulum ; v, virions; w, cell wall. (From Kitajima and Lauritis, 1969.)

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occur during replication. I n terms of genome size one can be relatively sure that these viruses represent one of the simplest types of autonomous DNA viruses, since their genetic repository is near the lower end of the scale of genome size among DNA viruses. Very likely the events during replication are similar to other DNA viruses of comparable genome size. At least six proteins, or gene products, would appear to be needed to account for the various functions one could postulate as essential for caulimovirus replication and dissemination in nature, viz., the matrix protein of the virus-specified inclusion body, four capsid proteins, and a t least one vector acquisition factor. Several other functions may be hypothesized, such as a virus-specific DNA polymerase for DNA replication, plasmodesmata transformation factors, and perhaps other viruscoded gene products needed for cell-to-cell movement. When one considers the various functions that are virus specified, there is a distinct possibility that caulimoviruses possess a split genome. I n fact, the genome size of 4 X lo6 daltons, specifying a messenger RNA of 2 X lo6 daltons, is not adequate to code for the four capsid polypeptides. Yet it is obvious the viruses specify other functions, e.g., the vector acquisition factor. Some, or perhaps all, of the caulimoviruses are very difficult to degrade chemically. As a consequence one wonders how uncoating occurs in the host cell. It is difficult to visualize uncoating as a spontaneous process in viruses so recalcitrant to chemical degradation. Proteolytic processes may be involved in release of the virus genome within the host cell, or a conformational change may take place in capsid polypeptides when the virus passes through the cell membrane that is effective in spontaneous release of the viral genome. After host cell entry and shedding of coat protein, the viral genome probably becomes associated with host RNA polymerase for its initial transcription to yield messenger RNA. Several attempts have been made, under a variety of conditions, to demonstrate RNA polymerase activity in virions of CaMV with negative results. Hence, in the absence of any evidence to the contrary, one can assume that host RNA polymerase participates in initial transcription. This is not unusual for small DNA viruses. A variety of small DNA phages and animal viruses are dependent on host RNA polymerase for transcription. As mentioned later, RNA polymerase may have a priming role in DNA replication also. A sequence of transcriptional events may occur in the production of messenger RNA. Based on information with various animal and bacterial viruses i t appears that sequential transcription is one of the chief control mechanisms for gene expression. With SV40, a virus with a DNA genome size slightly smaller than that of caulimoviruses, the viral genome consists of early and late functions. Only one of the two DNA strands is

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transcribed early in infection, whereas both strands are partially transcribed late in infection (Khoury et al., 1972; Sambrook et al., 1972; Lindstrom and Dulbecco, 1972). Thus portions of both strands have a messenger function. The early genes are transcribed before viral DNA replication. After the onset of viral DNA replication both early and late genes are transcribed. Early transcription occupies a template consisting of about one-third of one strand while the late template occupies about two-thirds of the opposite strand. With some larger DNA viruses early functions are transcribed by host cell RNA polymerase and late functions by a virus-specified enzyme synthesized as one of the early gene products. The intracellular site for initial transcription of caulimoviruses is not known. After transcription, however, as judged by observations with the electron microscope, initial translation probably occurs in the cytoplasm to produce the matrix protein of the inclusion body, although one cannot rule out nuclear involvement. Matrix material has a conspicuous appearance in stained tissue sections because of its unusual electron density, and i t can be easily distinguished before the appearance of virus (Fig. 8 ) . These electron dense patches (protoinclusions) are surrounded by ribosomes leading one to surmise that these are sites of intensive protein synthesis (Fig. 8). These protoinclusions grow by accretion and perhaps fusion to produce typical inclusion bodies in which the virions are synthesized. There is little doubt that the cytoplasmic inclusion bodies are the main sites for DNA replication and virus assembly. Both the sequential development of matrix material and later virions and autoradiographic evidence support this view. Autoradiographic evidence with tritiated thymidine administered to mature cells of Brassica infected with CaMV, which normally exhibit no DNA metabolism, show that viral inclusion bodies are active sites for the uptake of the thymidine label (Kamei et al., 1969; Fujisawa et al., 1971, 1972; Favali et al., 1973) and hence are loci for DNA replication. The restricted distribution of virus within the cell and the early formation of virions in the inclusion body indicate that these are the sites for virus assembly as well. DNA replication is a much more complicated affair than RNA synthesis and i t seems almost certain that viruses of this sort are dependent on host functions for replication of their genomes. For example, a multiplicity of enzymes is involved and an RNA primer molecule is required to initiate the process. DNA polymerase is only one component of the multienzyme complex involved in the replication of DNA. As a consequence, probably all small DNA viruses rely largely or wholly on hostspecified functions for replication of their DNA chromosome. Among the 20 odd DNA polymerase enzymes of various origin isolated

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to date, none can initiate a polynucleotide chain in vitro. This is a distinctive feature of these nucleotide joining enzymes. I n contrast, the RNA polymerases can initiate chains and do so a t specific nucleotide sequences. The DNA polymerases on the other hand are blind to initiation sequences, even a t the origin of a replication site, and instead have an obligate requirement for an RNA primer in situ on the template for initiation to occur (Schekman et al., 1974). Hence a separate stage of transcription precedes the subsequent stage of DNA synthesis (Brutlag et al., 1971). The RNA primer must have a free 3’-hydroxyl in order to react with an incoming 5’-nucleoside triphosphate, the substrates of DNA polymerase active in the elongation reaction. RNA polymerase, in some cases, is active in providing the needed RNA primer. However, a variety of mechanisms are active in the genesis of RNA primers in E . coli cells, a feature that further complicates the replication of DNA (Schekman et al., 1974). Moreover, multiple forms of DNA polymerase occur in cells; for example, three types are recognized in E. coli and five in mammalian cells. Smaller DNA viruses, probably wholly dependent on host-specified functions for replication of the viral genome, contrast sharply with small RNA viruses, which almost invariably specify at least part of an RNA polymerase active in replication of their own genome. Various viruses and plasmid DNAs have been isolated with the DNA primer still in place (Speyer et al., 1972) ; Williams et aZ., 1973; Rosenkranz, 1973). The recent discovery of RNA covalently attached to DNA of CaMV is probably another case of an RNA primer remaining in situ on a DNA template. With some DNA viruses that have a circular chromosome, the RNA primer is removed enzymatically before the DNA circle is closed. Some evidence indicates that the nucleus may have a role in the early stages of replication of the DNA of caulimoviruses although the main site of DNA replication is the cytoplasm. I n a recent report Favali et al. (1973), using a quantitative autoradiographic technique with tritiated thymidine with CaMV-infected plants, presented evidence for a significant increase in labeling of the nucleus in recently infected tissue, suggesting a nuclear site for the early stages of viral DNA replication. Some small DNA viruses of animals, such as polyoma and SV40, replicate in the nucleus. The larger DNA viruses of animals, such as the pox viruses, replicate mainly in the cytoplasm in large inclusion bodies termed “virus factories” from their role in synthesis and assembly. Although formerly these viruses were believed to be altogether independent of any nuclear function, some evidence (Walen, 1971) favors an association of viral DNA with the host chromosomes followed by the formation of sev-

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era1 intranuclear foci of virus replication. These foci then move into the perinuclear space and become typical cytoplasmic “virus factories.” A similar participation of the nucleus in the early stages of caulimovirus replication should be investigated. VII. CONCLUDING REMARKS

As yet we know relatively little about the intrinisic properties and replication of this interesting group of plant viruses. Interest in the group is increasing, however, as awareness develops of the advantages these viruses offer as a model system for studies of transcription, translation and DNA replication in plant cells. The caulimoviruses are the smallest independently replicating DNAs known a t the present time in plant cells. Moreover, they provide the only available examples of simple DNAs whose gene products are known and easily recognized, an important advantage for studies of transcription and t,ranslation. Hence, i t appears certain that these viruses will play an important role in molecular biological investigations with plant cells. ACKNOWLEDGMENTS The author is indebted to E. W. Kitajima, N. W. Frasier, A. A. Brunt, and R. H. Lawson for providing illustrations and to T. A. S h a h and L. J. Petersen for fixation and electron microscopy of isolated viral inclusions.

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