J . Cell Sci. Suppl. 7, 95-107 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987

95

STRUCTURE AND REPLICATION OF GEMINIVIRUS GENOMES

J. W. D A V I E S , J. S T A N L E Y , J. D O N S O N , P. M. M U L L I N E A U X a n d M. I. B O U L T O NDepai'tment of Vims Research, John Innes Institute, Colney Lane, Norwich NR4 7UH, UK

S U M M A R Y

The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.

I N T R O D U C T I O N

The geminiviruses appear to have twinned particles, about 20x35 nm, comprising a dimer of quasi-icosahedral units. Some members of the group can also form trimers and tetramers, but geminate structures are most common and are typical of the group. Each particle contains a small circular ssD N A molecule of between 2-5-3 ‘Okb in size, depending on the individual virus (for reviews see Francki et al. 1985; Harrison, 1985; Davies et al. 1987).

The members of the geminivirus group can be sub-divided on the basis of host range (monocotyledonous or dicotyledonous species) and insect vector (leafhopper or whitefly species). We have studied cassava latent virus (CLV) which is whitefly- transmitted and infects dicotyledons, includingN icotiana species: maize streak virus (M SV) which is leafhopper-transmitted and as its name implies infects monocoty­ledonous hosts which in addition to maize include rice, wheat, sugarcane, sorghum, millets and grasses: and a sort of intermediate, beet curly top virus (BC TV ) which is also leafhopper-transmitted but experimentally can infect a wide range of dicoty­ledonous but not monocotyledonous plants (Bennett, 1971).

G E N O M E S T R U C T U R E A N D O R G A N I Z A T I O N

CLV was the first geminivirus to be sequenced (Stanley & Gay, 1983). The sequence data revealed two different D N A circles, which are called arbitrarily DNAs 1 and 2. The virion-encapsidated D N A was designated the ( + ) sense. This does not mean to imply that its complementary or ( —) sense DNA is non-sense: indeed both senses carry open reading frames (Fig. 1). Other whitefly-transmitted dicotyledon- infecting geminiviruses, tomato golden mosaic virus (TG M V ) and bean golden

mosaic virus (BGMV) have a similar genome organization (Ham ilton et al. 1984; Howarth et al. 1985). Comparisons between these three viruses (Hamilton et al. 1984; Townsend et al. 1985; Howarth et al. 1985) plus the analysis of CLV RNA transcripts (Townsend et al. 1985), suggests a maximum of six genes. Of these the coat protein gene of CLV was identified as the 30-2K O RF on D N A 14-.

96 J . tr . Davies, J . Stanley, J . Donson, P. M. Mullineaux and M. I. Boulton

Fig. 1. Organization of the genome of CLV. The orientation and coding capacities (in kDa) of ORFs with homologous counterparts in other whitefly-transmitted geminiviruses are shown. Intergenic sequences common to both DNAs 1 and 2 are boxed. The major transcripts are indicated on the outside of each map.

Structure and replication of geminivirus genomes 97

Fig. 2. Organization of the genome of MSV. The orientation and coding capacities (in kDa) of ORFs are given. Two non-coding regions are indicated (stippled). Solid arrows (♦) are regions of potential secondary structure discussed in the text. The major transcripts are shown on the outside of the map.

T he nucleotide sequences of CLV DNAs 1 and 2 are very different, except for a region of about 200 nucleotides, which is almost identical on each DNA. This common region is non-coding, though it may have a regulatory function in replication and /o r expression common to both DNAs.

Stanley (1983) dem onstrated by molecular cloning that both DNA components were required for plant infection, thus dem onstrating the true bipartite nature of the genome; which implies that two different virus particles are necessary for infection of plants. Excised cloned CLV DNA is very infectious (Stanley, 1983) even as a linear ds molecule, implying that in vivo circularization can occur, and suggesting that ds DNA may be a true replication intermediate. Given this, it therefore came as a surprise that the first sequence of a geminivirus infecting monocotyledonous plants and being leafhopper-transm itted, namely MSV, seemed to have only one DNA circle. All of the sequence data fitted in one circle (Fig. 2; Mullineaux et al. 1984). Recently, experiments in collaboration with N . Grimsley, B. Hohn and T . Hohn in Basel have shown that a dimer construct of this MSV DNA in a T i plasmid, can ‘agroinfect’ maize plants. T he normal pattern of MSV infection was seen after inoculation with the Agrobacterium construct (Grimsley el al. 1987). T his strongly suggested that MSV had a monopartite genome of 2687 nucleotides, and therefore the smallest known viral genome.

T he MSV sequence is very different to CLV; indeed, there is no detectable homology at the D NA level, though there is some homology at the amino acid level (M ullineaux et al. 1985). Some limited familial homology is found between the coat proteins of the geminiviruses. Flowever, it is the non-structural proteins, some of

98 J . W. Davies, jf. Stanley, jf. Donson, P. M. Mullineaux and M. I. Boulton

Fig. 3. Organization of the genome of BCTV. The orientation and coding capacities (in kDa) of ORFs with counterparts in other geminiviruses with docotyledonous host ranges are shown. The position and orientation of possible promoter (open triangles) and polyadenvlation signals (closed triangles) are indicated.

which may be involved in the replication of the viral genome, which are most relevant to this report.

S trong familial and some direct homology between the putative ( —) sense products of geminiviruses (DNA 1 of whitefly-transmitted members) (M ullineaux et al. 1985; MacDowell et al. 1985; Stanley et al. 1986) suggest that these ORFs m ight code for proteins central to their life cycle, such as those involved in the DNA replication.

Beet curly top virus (BCTV) DNA has recently been sequenced (Stanley et al. 1986) revealing only one circle (Fig. 3). BCTV can be mechanically transm itted by pin-pricking inocula into the crowns of young Beta vulgaris, and it was an infectious clone that was sequenced. It is therefore, as suggested for MSV and WDV (wheat dwarf virus, McDowell et al. 1985), m onopartite. However with the exception of the coat protein O RF, the BCTV genome shows strong homologies with D N A 1 of other geminiviruses, such as CLV which infect dicotyledonous plants.

Transcription of CLV and MSV has been shown to be bidirectional (Townsend et al. 1985; M orris-K rsinich et al. 1985). Transcripts have been mapped that account for each of the six ORFs of CLV predicted from sequencing data (Townsend et al. 1985). For CLV, in addition to the coat protein gene on DNA 1( + ), in vitro translation of ( —) sense CLV RNA transcripts identified products of 40K and 15K mol. wt. (Fig. 4).

T he larger CLV transcripts are initiated on either side of the IR (intergenic region) common to both DNAs. All transcripts term inate at a point diagonally opposite this,

Structure and replication of geminivirus genomes 99

there being a very small intergenic region here comprising only a few nucleotides, within an extremely A /T rich environment. In CLV and related viruses, the putative polyadenylation signal AATAAA is often present within this region.

T his type of bidirectional arrangement is reminiscent of some animal DNA viruses, such as polyoma and SV40 where the genome is divided into early and late genes which are expressed before and after the initiation of DNA synthesis. T he structural genes of geminiviruses (e.g. the capsid proteins) are expected to be late functions. By analogy, the opposite strand ORFs for the 40K and 15K products may be early functions. However, there is no direct evidence for temporal control of geminivirus genomes.

For MSV two large (1-05 and 0-9kb) RNAs, overlapping, on the coat protein gene (27K O R F) strand (4- sense) have been mapped (M orris-Krsinich et al. 1985); the

4 0 k

3 0 k

Fig. 4. In vitro translation of CLV ( —) sense polyadenylated RNA. Total polyadenylated RNA from infected and healthy AT. benthamiana plants was selected with ( + ) sense virion DNA to purify ( —) sense sequences. In zvtro translation was carried out in a wheat germ extract, labelling with [ S ] methionine. Approximate sizes were estimated by reference to [l4C]protein markers and other viral translation products (not show'n). I = infected, H = healthy, C = control with no added RNA. Virus-specific products of ~40 K , ~15K and possibly ~30K and ~13K are compatible with the putative CLV ( —) sense products (J. W. Davies & D. Robinson, unpublished results).

100 jf. W. Davies, J. Stanley, J . Donson, P. M. Mullineaux and M. I. Boulton

1-05 kb RNA overlaps the 10-9K O RF in addition to the 27K O RF. We have recently demonstrated that the 10-9K polypeptide is synthesised in vitro from a mixture of RNAs, and is present in plants cells, by using an antiserum to a 10-9K-/3- galactosidase fusion product expressed in E. coli (van der Vlugt, Mullineaux & Davies, unpublished). The function of the 10-9 K product has not been determined. It is not known which of the two RNAs of 1-05 and 0 '9kb is involved in the expression of the coat protein or whether the 1-05 kb RNA is translated as a dicistronic messenger.

On the ( —) sense, so far only one large RNA (l-2kb) has been detected, which maps to the 31K and 17K O RFs. How and if these two O RFs are expressed is not yet clear. Homologies between them and the N H 2 and COOH terminal amino acid sequences of 40—41K mol. wt. putative products from the (—) sense of DNA-1 of geminiviruses infecting dicotyledonous plants (CLV, TG M V , BGM V and BCTV) might suggest that these two coding regions are expressed as a single composite protein (Howell, 1984; MacDowell et al. 1985; Mullineaux et al. 1985).

There is a large intergenic region (L IR ) in an equivalent position to the 200 nucleotide common region of CLV, and diagonally opposite, a smaller one (S IR ). The transcripts terminate at sequences flanking the SIR .

R E P L I C A T I O N

We have discussed above mainly structural features of the geminivirus genomes, some of which may be relevant to understanding replication. In order to ask the right questions, it is necessary to have some clues about what might be involved in replication. This would at least facilitate the design of future experiments. Several observations are worth mentioning. First, geminiviruses are generally found in the nuclei, and in the case of M SV, large numbers of virus particles accumulate and aggregate there, and are easily seen. On closer inspection, we see paracrystalline arrays of virus-like particles (Francki et al. 1985; Stanley & Davies, 1985). The nuclei become hypertrophic and separate into granular bodies and fibrous structures.

In BG M V or CLV infections, nuclear structures called fibrillar rings are seen (Francki et al. 1985; Stanley & Davies, 1985), the origin and function of which are not known. These vary in size, and are never tubular, so must actually be spherical in nature. The granular nucleolar structures are largely ribonucleoprotein, but these fibrillar rings are thought to be deoxynucleoprotein structures. It has been suggested that they may be the sites of D N A synthesis (Kim et al. 1978). However, such structures have been observed in apparently healthy plants, albeit, when under some sort of stress.

For several whitefly-transmitted geminiviruses, the deproteinised viral ssD N A has been shown to be infectious (Goodman, 1977; Hamilton et al. 1981; Ikegami et al.1984). It is likely that the first step in D N A replication is second-strand D N A synthesis by host-encoded nuclear enzymes. Subsequent replication of the dsD N A (R F) may involve viral-coded products. Do we have any clues as to the whereabouts of the initiation site(s) for second-strand synthesis and the origin of dsDNA

Structure and replication of geminivirus genomes 101

c -C L V G A A C AC C C A A G G GG C C A A C C G - T A T A A T A T T A C C G G T T G G C C C C GC CCC T T T

• • • • • • • • • ̂ ^BGMV C A T A C A C G T G G C G G C C A T C C G A T A T A A T A T T A C C G G AT G G C C G C C C G C G CC C C T

TGMV G G G G C A C G T GG C G G C C A T C C G T T T A A T A T T A C C G G AT GG C C G CG C G AT CG T C™ • • • • • • • • • ^

BCTV A A C T T T C A T A A G G G C C A T C C G T T A T A A T A T T A C C G G AT G G C C C GAAAAAAATGG• • • • • • • • •

MSV C C A GCA GGAAAAGAAGGCGCGC A C T A A T A T T A C C G C G C C T T C T T T T C C T G C GAG• • • • • • • • •

WDV G T C G G G GGG CCT C C A C G C G G G T T A T A A T A T T A C C C C G C G T G G T G G C C C C C GACG

DSV G C C G C GGG GGG TG GGG CG C A C C A C T A A T A T T A C A G C C C C A C C C C C T G C G AGCCC

Fig. 5. Sequences adjacent to the nanonucleotide TAATATTAC, conserved in all geminiviruses so far examined. Complementary sequences are underlined. Alternative nucleotides found in DNAs 1 and 2 of CLV and TGM V are indicated.

replication? Fig. 1 shows the homologous region within the intergenic region of CLV D N A . Functions common to both CLV D N As are packaging and replication. Consequently, these homologous regions are likely to contain the origin of replication (OR) and/or a priming site for second-strand synthesis. Included in these regions are GC-rieh complementary sequences, flanking an AT-rich sequence. Very similar stretches are found in other geminiviruses, as shown in Fig. 5. In fact, the nanonucleotide TAATATTAC is conserved in all geminiviruses examined so far.

The possible involvement of such potential stem-loop structures in replication has been referred to in relation to other geminiviruses (Francki et al. 1985; Harrison,1985). An analysis of S I nuclease sensitive sites of TG M V dsD N A suggested the occurrence of a looped-out cruciform structure (Sunter et al. 1984).

There are nine possible stable stem-loop sequences in M SV DNA with free energy values greater than — 14kcalmol_1 (Mullineaux et al. 1984). Three of these are adjacent within the L IR and include the one which has the AT-rich sequence common to other geminiviruses (Fig. 5). Although two others are near the S IR of M SV D N A , these are not conserved in the closely related digitaria streak virus (D S V ; Dollet et al. 1986; Donson et al. in preparation). For both M SV and D SV a population of small D N A molecules, approximately 80 nucleotides in length and complementary to part of the SIR , are found associated with virus particles (Fig. 6). These molecules have ribonucleotides covalently linked to the 5' terminus of the D N A moiety. These molecules can prime second-strand synthesis in vitro , produc­ing double-stranded D N A that is apparently full-length and which on subsequent cloning is infectious to plants (Donson et al. 1984; Donson et al. manuscript in preparation). This priming event occurs at one position on the genome, at the 5' terminus of the population of small DNA molecules. The ribonucleotides covalently linked to the 5' terminus of the small D N A molecules are analogous to RNA primers for D N A synthesis found in a variety of prokaryotic and eukaryotic systems (Kornberg, 1980).

D N A secondary structure is known to be involved in the initiation of second- strand synthesis in D N A phages (Kornberg, 1980). Of that found between the related geminiviruses D SV , M SV and WDV, only the structure illustrated in Fig. 5 is conserved in position. Such a structure could act as the site of assembly for

102 y. W. Davies, J . Stanley, y. Donson, P. M. Mullineaux and M. I. Boulton

________16-9k_______ _ (A S L D D A P A # 5 r U T T T T T T T T A A T T T T A T G T A T C G C A G G G T G A C

3' C G A A G C G A G C T G C T G C G C G G C C G C A T T A A G T C T A G A C A C A A A A A A A A A ’T T A A A A T A C A T A G C G T C C C A C T G

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

1 2 4 0 1 2 30 1 22 0 1 2 1 0 1 20 0 1 19 0 1180

C C A G C G A G C C G G T C T T A A G C C C G G C G C GG G G T C A TG T GT GT TT T A G T T 3 G G T C G C T C G G C C A G A A T T C G G G C C G C GC CC C A G T A C A C A C A A A A T CA A C G G C GA AC 5'

+ +++++ + t++ +++ + + ++++++++++ ++++1 1 7 0 1 1 60 1 15 0 1 14 0 1 13 0 1 1 2 0

Fig. 6. Nucleotide sequence of the small DNA molecules associated with DSV. The sequence is shown aligned with the viral strand sequence. All the bases are deoxyribo­nucleotides except for the uracil residue and indeterminate numbers of undefined ribo­nucleotides (r) at the S' terminus of the molecules. The four most predominant 3' termini ( followed by ) are indicated. Sequences conserved with MSV are indicated (+) . Alignment of DSV and MSV sequences for the homology shown required the insertion of five padding characters ( ) in the DSV sequence at position 1161 on the virion sense strand. The position of the stop codon (# ) and the single letter codes of the eight carboxy terminal amino acids of the putative (16'9K) ( —) sense product are given (Academic Press).

proteins involved in second-strand synthesis. By analogy with 0X 174 (Arai & Kornberg, 1981), the resulting primosome could then direct primer synthesis elsewhere on the genome such as the SIR .

Fig. 6 shows sequences conserved between M SV and D SV in the region of the small D N A molecules. The involvement of these as well as encapsidation on the initiation and termination of the synthesis of the RNA and D N A moieties of the small molecules awaits further experimentation. Any mechanism will have to account for CLV not having a similar population of small DNA molecules associated with its virions (Stanley & Townsend, 1985).

So far we have discussed mainly the ssD N A , and the small primer-like D N A , but have only briefly eluded to other DNA forms which are found in virions, infected plants, and protoplasts. Clues to the replication mechanism are suggested by examination of these forms of DNA.

In virions (ssD N A ) and infected plants (dsDNA) both M SV and CLV DNA is found not only as ‘unit length’ circles, but also as multimers, which are mainly dimeric (Stanley & Townsend, 1985). Any proposed replication model must therefore account for dimers, at least, and a rolling circle mechanism is a possible explanation. There are also D N A s smaller than unit length (‘minicircles’) which are usually about half-sized circles. These defectives occur only as sub full-length species of D N A 2 (Stanley & Townsend, 1985). Several of these CLV D N A 2 defective molecules have been cloned and analysed. The deletions are always from just downstream of the common region, to a point within the 3' end of the 33-7K O RF (Fig. 7). Small sequences ranging from di- to hepta-nucleotides, repeated at the point of deletion, may be responsible for locating the limitation of the deletions. The deletions never include the common region supporting the suggestion that this has a

Structure and replication of geminivirus genomes 103

replication function. Similar structures have since been found for TG M V D N A 2 (MacDowell et al. 1986).

T o follow the kinetics of the production of the various DNA forms, to do pulse- labelling or carry out m utation studies to locate the origin of replication, we need a synchronous culture system. Such experiments cannot be done with whole plants. Recently, Townsend et al. (1986) have managed to infect Nicotiana plumbaginifolia protoplasts with CLV, and cloned CLV DNA. A truly synchronous culture has still not been achieved, but these preliminary experiments reveal two more im portant clues concerning replication.

Fig. 8A shows the result of infecting the protoplasts with whole virus (CLV). The virion D N A is single-stranded, but both ds (as cccDNA) and ssDNA progeny appear. From this we can conclude that both DNAs (1 and 2) are replicating. Synthesis of D N A is only evident from Day 2 onwards. Perhaps significantly, Day 2 is the time at which cell-division began. Viral DNA replication may be synchronized with host D N A replication, or has a prerequisite for host DNA replication or cell division.

A similar experiment, also shown in Fig. 8, was performed with excised cloned CLV DNAs 1 and 2. When both DNAs were present in the inoculum, progeny viral DNAs similar to those observed using a virus inoculum were synthesised, which again became apparent after Day 2. A similar result was obtained when cloned DNA

Fig. 7. CLV DNA 2 showing the extent of the nucleotide deletions found in molecules extracted from CLV infected plant tissue. The deletions are shown relative to the region common to DNAs 1 and 2 (stippled area) and the major open reading frames with potential coding capacities of 33-7 and 29-3 kilodaltons. Deletions 1 to 4 correspond to the four classes of deletion characterised by Stanley and Townsend (1985) ( IRL Press (Oxford)).

a < I n o c u l u m -<r Z^ 0 p a r t i a l l y p u r i f i e d v i r u s

104 J. W. Davies, J . Stanley, J . Donson, P. M. Midline aux and M. I. Boulton

T- OJ < <

Û Coo —2 z o ±Q û o > O T- CM CO ' t U)

T> T3 "O T3 T3O i - C\J (0 ID■O 'O T3 TJ T3 TJ

p r o b e D N A 1 p r o b e D N A 2Fig. 8. Replication of CLV DNA in Nicotiana plumbaginifolia protoplasts after inocu­lation with virus (A) or cloned DNAs (B). Samples were taken daily (d0-d5) from protoplasts inoculated over a five day period. The positions of linear dsDNA (lin ds), cccDNA and circular ssDNA (css) are indicated. The gradient-purified linearised cloned DNA inocula can be seen to contain residual amounts of the linearised M l3 cloning vector. (Photograph courtesy of R. Townsend.)

1 alone was used as the inoculum. However, when D N A 2 alone was used, no viral D N A replication could be detected (Fig. 8B). In whole plants, both CLV DNAs are required for a systemic infection: the genome is bipartite in this respect. In protoplasts, it has been dem onstrated that DNA 1 but not DNA 2 can autonomously replicate. T his leads us to conclude that DNA 2 carries a cell-to-cell spread function. T hat DN A 1 carries all that is required for DNA replication is consistent with the prediction that the 40K and /o r 15K products may have a replication function, and that the single circle genome viruses, MSV, WDV and BCTV are autonomously replicating ‘DNA 2-minus’ genomes.

In summary, we believe that replication occurs in the nucleus where virus and cytopathological structures are found. T here is some evidence that viral DNA synthesis is synchronised with host DNA replication. Conversion of ssDNA from the infecting virus to cccDNA probably utilizes nuclear enzymes. T he multimeric forms

Structure and replication o f geminivirus genomes 105

I n o c u l u m -

D N A s 1 &2 D N A 1 D N A 2' • ' ‘ ' ----- -

o c o ^ ' n o c o r i L o o c o ' ^ - i nT J ' D ' D ' O ' D T J T J D ' D ' O ' D ' a

I i n d s

c c c

c s s

P r o b e D N A 1 • V2 s a m p l e l o a d e d

of ss and dsD N A suggest that conversion of cccDNA to cccDNA, and cccDNA to ssDNA might proceed via a rolling circle mechanism.

A series of experiments designed to expand our knowledge of geminivirus DNA replication are in progress. These include-the identification of genes and control sequences involved in replication, by deletion and site-directed mutagenesis studies and by the study of m utant genomes in whole plants and protoplasts. We also anticipate that constructs with marker genes, designed to analyse prom oter activity, will provide valuable data concerning DNA replication.

We thank Dr R. Townsend and other colleagues in the Virus Research Department for many interesting discussions, and Muriel Hobbs for typing the manuscript. This work is supported by a grant-in-aid from the AFRC to the John Innes Institute, and by Agrigenetics Advanced Science Co. (Lubrizol Genetics, Inc.). The work was carried out under MAFF licence nos. PHF 49 A/85 (17) and pH F 49 A/41 (126).

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