y . Cell Sci. Suppl. 7, 00-00 (1987)
Printed in Great B ritain © The Company o f Biologists Lim ited 1987
213
ST R U C TU R E AND REPLICATION OF CAULIMOVIRUS
GENOMES
R. H U L L , S. N. C O V E Y
and
A. J . M A U L E
Joh n Innes Institute, Colney Lane, Norwich NR4 7UH, UK
SUMMARY
In this paper the current state of knowledge of the replication of cauliflower mosaic virus
(CaMV) is reviewed and the DNA intermediates and enzymes involved in replication are
discussed. Based on this information a model for the replication complex is developed. In this
model it is suggested that replication complexes resemble virus particles and that, in their
assembly, there are close interactions between the inclusion body protein, the virus coat protein,
the replicase enzyme, the tRN A primer and the 35S RNA template. T he similarities between
CaMV replication complexes and those of retroviruses are discussed, and we extend this discussion
to a comparison between CaMV and reverse transcribing elements.
INTRO DUCTION
Caulimoviruses are the only group of plant viruses whose particles are known to
contain double-stranded DNA. Among the other morphological and cytological
features which characterize members of this group are isometric particles which are
strongly stabilized and which are usually found only within virus-specific, cytoplas­
mic proteinaceous inclusion bodies. The caulimovirus group has recently been
reviewed by Hull (1984).
Most of the information on the molecular biology of caulimoviruses comes from
studies on the type member of the group, cauliflower mosaic virus (CaMV) (for
general reviews see Covey, 1985; Maule, 1985a; Hull & Covey, 1985; Covey & Hull,
1985; Hohn et al. 1985). T o introduce this chapter we will described the salient
points of caulimovirus molecular biology. Caulimovirus virion DNA is a double­
stranded circular molecule of about 8kbp containing gaps or discontinuities at
specific sites. There is always one strand with one discontinuity (gap 1) which, for
CaMV, is the transcribed or ( —) strand. The transcripts have the potential to code
for 6 or possibly 8 proteins larger than lOkDa; in carnation etched ring virus
(CER V ) there are 6 long open reading frames (Hull et al. 1986). The complementary
strand has either one, two or three discontinuities depending upon virus or strain of
virus (Hull & Howell, 1978; Hull & Donson, 1982; Donson & Hull, 1983; Richins &
Shepherd, 1983). At each discontinuity, at least for CaMV, the 5' deoxyribonucleotide is at a fixed position (Hull et al. 1979) and frequently has one or more
ribonucleotides attached (Guilley et al. 1983); the 3' terminus extends beyond the
5' terminus by a variable amount (5 -4 0 nucleotides) giving an apparently triple­
stranded structure (Francki et al. 1980; Richards et al. 1981). RNA is transcribed
from a supercoiled form of DNA which associates with host proteins (probably
214
R. Hull, S. N. Covey a n d A. J . M aule
histones) to produce a mini-chromosome (Olszewski et al. 1982). There are two
major RN A transcripts, the 19S RNA which is the mRNA for the major protein of
inclusion bodies (the gene VI product) and the 35S RNA which covers the full
genome and has a terminal repeat of 180 nucleotides (see reviews cited above).
• T he recognition that the CaMV genome has several sequences and organizational
features in common with retroviruses, together with the realization that members of
another DNA virus group, the hepadnaviruses, involved reverse transcription in
their replication (Summers & Mason, 1982) led to the development of a detailed
model for CaMV replication (Hull & Covey, 1983a; Pfeiffer & Hohn, 1983). Since
then the model has continually been refined. The first phase of the replication cycle is
in the nucleus where the input D N A, having been covalently closed by a mechanism
not yet determined, is transcribed by host RNA polymerase II (Guilfoyle, 1980) to
give the 19S and 35S RNAs. The 19S RNA is translated in the cytoplasm to produce
the inclusion body protein. The 35S RNA forms the template for the reverse
transcription phase of replication which is considered to take place in replication
complexes in association with the inclusion bodies. Priming of the ( —) strand DNA
synthesis is by plant cytoplasmic tRN A met initiator adjacent to the site that will form
gap 1 at about 600 nt from the 5' end of the template. The formation of strong-stop
DNA (termed sa-D N A ; see below), the first strand switch, the formation of ( + )
strand primers at purine rich regions (adjacent to the sites of gaps in the plus strand)
and the second strand switch are considered to be by mechanisms similar to those of
retrovirus replication. The main difference with retrovirus replication arises when
the oncoming newly synthesized strand reaches a priming site. In CaMV it appears
that only limited strand displacement takes place thus giving the characteristic
structures of the gaps. This completes the mature virion DNA.
In this chapter we are going to discuss the nature of replicative intermediates
which are important to the elucidation of the replication mechanism, the structure
and functioning of the replication complexes, and we will draw attention to the
similarities and differences between the replication complexes and retrovirus
particles. We will then expand the comparison with retroviruses to include
hepadnaviruses and retrotransposons.
R EV ERSE TRA N SCRIPTION REPLICATION IN TERM ED IA TES
One approach to understanding details of the CaMV replicative process has been
to analyse the structure of viral DNA forms isolated from infected leaves or from
protoplasts infected in vitro. T h ere are two basic types of replication intermediates,
those which are extractable by phenol alone and those which resemble encapsidated
DNA in that they are extractable by phenol only after prior protease treatment. A
variety of viral DNA forms are found in phenol-extracted tissue (Hull & Covey,
19836). These forms have been variously termed ‘intracellular’, ‘unencapsidated’ or
‘free’ DNAs and this fraction contains relatively little virion D NA. A fundamental
feature of reverse transcription as a means of generating double-stranded DNA from
single-stranded RNA is that it proceeds in two phases. First, a ( —) strand DNA copy
Structure a n d replication o f caulim ovirus genomes
215
of the RN A template is made and second, this D NA phase becomes the template for
( + ) strand synthesis. Thus, ( + ) strand synthesis cannot begin until after ( —) strand
synthesis has started and passed at least the first priming site. From this it follows
that, in theory, it should be possible to detect ( —) strands as single-stranded DNAs
whilst ( + ) strands exist only in a double-stranded form in cells.
One of the first CaMV replication intermediates to be characterised was a small
fragment of viral DNA some 600 nucleotides long that co-purified with poly (A) +
RNA and was termed sa-DNA (Covey et al. 1983). Using strand-specific probes-, it
was shown that sa-DNA was of ( —) strand polarity and it also mapped to a region of
the CaMV genome between the 5' terminus of 35S RNA and the position of the ( —)
strand gap (G l) in virion DNA. Some of the sa-DNA molecules appeared to be base
paired with two smaller fragments of ( + ) strand DNA whilst others were apparently
single-stranded.
An unusual feature of sa-DNA was that an RNA molecule approximately 75
nucleotides long (Covey et al. 1983) was covalently attached to its 5' end and this
fragment had characteristics of plant cytoplasmic tRNAmet initiator (Turner &
Covey, 1984). The structure of sa-DNA was reminiscent of retrovirus ( —) strand
‘strong-stop’ DNA, a very early reverse transcript of the genomic RNA 5' end that
originates at the primer binding site (pbs ). It seems very likely, therefore, that saD N A is the CaMV version of this strong-stop DNA. Appreciable amounts of saD N A were found associated with CaMV virions in a DNase-resistant form (Turner
& Covey, 1984) and this raises the possibility that sa-DNA itself becomes a primer
for reverse transcription and accumulates in excess to overcome any pause caused by
the first template switch at the termini of 35S RNA. The nature and function of the
association of sa-DNA with virions is not yet understood although curiously it is not
fully protected from nuclease attack since the RNA moiety was found to be
susceptible to RNase (Maule, 19856).
Single-stranded DNAs of ( —) sense polarity larger than sa-DNA have also been
reported. Marsh et al. (1985) isolated heterogeneous ( —) strands, that ranged in size
between 0 - 6 kb and 8 - 0 kb, from CaMV putative replication complexes. They also
found that ( + ) strands existed only in a duplex form with ( —) strands. Hull & Covey
(19836) had previously demonstrated the presence in DNA extracts of infected leaves
of double-stranded CaMV DNAs with single-strand extensions of ( —) strands; no
single-stranded ( + ) strands were observed. Thomas et al. (1985) showed that
isolated CaMV replication complexes were capable of synthesizing both ( —) and ( + )
strands and that this synthesis was asymmetric. ( —) strands with a size range similar
to that reported by Marsh et al. (1985) were also observed by Thomas et al. (1985).
Thus, several lines of evidence show that CaMV replication exhibits asymmetry as
the reverse transcription scheme would suggest.
Less is known about the priming and elongation of CaMV ( + ) strands. It seems
clear that ( + ) strands originate close to the two ( + ) stand gaps (G 2 and G3) although
the presence of additional gaps in a sub-population of virion DMA molecules (Maule
& Thomas, 1985) suggests that there might be other ( + ) strand priming sites as well.
D NA ( + ) strand synthesis is probably primed by RNA oligomers since the remnants
216
R. Hull, S. N. Covey a n d A. J . M aule
of these putative primers have been detected in virion DNA by Guilley et al. (1983).
The findings of genome-length ( —) strands described above suggests that ( + ) strand
priming is a relatively slow event although ( —) strands might accumulate if ( + )
strand priming is prevented by loss of the RNA primers by excessive RNase H
activity. In this respect it is interesting to note that Covey & Turner (1986) have
discovered a population of double-stranded CaMV hairpin DNAs that originate at
the pbs and their structures suggest that synthesis of double-stranded DNA is
possible in the absence of a specific ( + ) strand priming event regulated by RNA
oligomers. Quite what function this phenomenon serves in the CaMV replication
cycle remains to be seen. Putative replication intermediates isolated from infected
plants are shown in Fig. 1.
THE R EV E R SE TRA N SCRIBIN G ENZYM E
Characterisation of the DNA polymerase in CaMV replication complexes
in relation to its role as a reverse transcriptase has been based on its specificity
for exogenously added artificial template and natural heteropolymeric RNAs as
template/primers, its requirement for monovalent and divalent cations, sensitivity to
actinomycin D, RNase and DNase, a correlation between the size of the active
protein and the theoretical size of the putative pol gene product and an antigenic
relationship between the primary pol amino acid sequence and a polypeptide present
in replication complexes (see Hohn et al. 1985). That CaMV O R F V product does in
fact provide the reverse transcriptase (pol) function is supported by amino acid
sequence homology comparisons between known reverse transcriptases and O R F V
product (Toh et al. 1983; Patarca & Haseltine, 1984; Volovitch al. 1984; Toh e ta l.
1985) and is proven by the demonstration that cloned O R F V is expressed in yeast to
give a functional enzyme (Takatsuji et al. 1986).
In common with other reverse transcriptases, the enzyme is capable of utilizing
poly rA/oligo d T as a template primer (Guilfoyle et al. 1983; Volovitch et al. 1984)
and is distinguishable from y-like polymerases in its utilization of poly rC/oligo dG
(Volovitch et al. 1984). The enzyme showed a requirement for Mg2+ (Pfeiffer et al.
1984; Thomas et al. 1985) and monovalent salt at 30-50m M (Pfeiffer et al: 1984;
Volovitch et al. 1984; Thomas et al. 1985).
Perhaps the most compelling experimental evidence in favour of the CaMVspecific polymerase being capable of reverse transcription has been its demonstrable
capacity to copy exogenously applied heteroribopolymers (Volovitch et al. 1984;
Thomas et al. 1985). Thomas et al. (1985) showed that the enzyme copied cowpea
mosaic virus RNA primed with oligo dT into cDNA of both ( —) and ( + ) polarity.
This reaction was dependent upon the replicase complex preparation being subjected
to a single freeze-thaw cycle, a treatment which presumably released the endogenous
template from the enzyme.
It can be predicted that replication by reverse transcription would be sensitive to
RNase but insensitive to actinomycin D for the synthesis of ( —) strands and sensitive
to both DNase and Actinomycin D for the synthesis of ( + ) strands. This is partially
Structure and replication of caulimovirus genomes
217
1
3
"
+
+2
5v □ — o—
------------ •------------------- •--------------------------- □ 3'
<^—o®1 O— o -
1
Fig. 1. Products of reverse transcription of C aM V genomic RN A . T h e top line
represents the genomic RN A showing the terminal repeat the ( —) strand priming site (O )
at gap 1 and the ( + ) strand priming sites (O ) at gaps 2 and 3. T h e next seven lines or pairs
of lines show the various form s of D N A intermediates which are transcribed from the
R N A which are described in Hull & Covey (19836), Covey et al. (1983), T u rn er & Covey
(1984) and Covey & T u rn er (1986). T h e genomic D N A is shown at the bottom .
substantiated by the published data but the experim ents are com plicated by the
dem onstration that nucleic acids in replication com plexes have significant resistance
to nucleases.
F o r th e C aM V D N A polym erase to act as a reverse transcriptase it would, by
necessity, require that an R N ase H activity be associated with it. W ith retroviruses
this fu n ction is carried out by a domain within th e p o l gene product w hich, from the
analyses of am ino acid sequence hom ologies (T o h et al. 1985; Hull et al. 1986),
would appear to exist also in the gene V product of C aM V and C E R V . T h e
m echanism by w hich R N ase H degrades the R N A tem plate after synthesis of ( —)
218
R. Hull, S. N. Covey a n d A. jf. M aule
DNA strands is less clear. Three reactions occurring during replication through
reverse transcription indicate that the RNase H should have at least some
endonucleolytic activity: firstly, the 35S RNA template must have the poly (A) tail
cleaved from its 3' end: secondly, the tRN A primer should be removed from the 5'
end of ( —) DNA after the pbs has been copied into ( —) strand D N A ; thirdly,
oligomeric RNA primers for ( + ) strand synthesis are most likely generated by
cutting 3 5 S RNA in hybrids with ( —) strand D NA. Accordingly, RNase H activity
has been shown to remove the tRNA primer from the natural substrates of the
retroviruses, avian myeloblastosis and murine leukaemia viruses, and to produce ( + )
strand primers in in vitro reactions (see Varmus & Swanstrom, 1982).
T he dogma of reverse transcription is that the RNA template is degraded in a
processive way after being transcribed into ( —) stand DNA, although evidence in
support of this for caulimoviruses is lacking. Similarly, the relationship between the
rate, direction and time of degradation mediated by RNase H and the growing ( —)
strand has also not yet been resolved for caulimoviruses although the observation that
preparations of replicative intermediates from CaMV-infected turnip plants contain
genome length R N A /D N A hybrids (Marsh et al. 1985) may point to a 5 ' —>3'
activity.
T he efficiency with which RNase H is able to generate ( + ) strand primers through
its failure to digest polypurine stretches of RNA appears to be variable in CaMV, as
preparations of virion DNA which contain a small proportion of molecules with
genome-length ( + ) strands (Maule & Thomas, 1985) and the detection of near
genome length hairpin molecules (Covey & Turner, 1986) both suggest that primers
have been lost, perhaps through an excessive appetite for the substrate.
Most studies of the in vitro CaMV polymerase have noted the marked asymmetry
in activity seen as an overproduction of ( —) strands in relation to ( + ) strands, also
recorded for many retrovirus polymerases. Whilst the model for CaMV DNA
replication through reverse transcription calls for a temporal asymmetry in synthesis,
this disparity leads us to question whether the conditions most appropriate for ( —)
strand synthesis in vitro are in fact suboptimal for ( + ) strand synthesis. Further­
more, we believe that the two processes are functions of separate subdomains of the
same or separate molecules. Analysis of amino acid homology between CaMV and
C ER V pol gene products has identified two distinct regions within the polymerase
domain which may represent the two activities (Hull et al. 1986). Alternative or
further explanations could be that in vitro and in vivo the two activities function at
very different rates and/or that the efficiency of priming for ( + ) strand synthesis is
low.
T he characteristic structure of caulimovirus virion DNA which has a single
discontinuity in the ( —) strand and, for most isolates, at least two discontinuities in
the ( + ) strand, conforms with the model for DNA replication in which synthesis is
continuous for the former and discontinuous for the latter. In this respect,
caulimoviruses are similar to avian retroviruses (Varmus & Swanstrom, 1982).
However, the existence of virion DNA molecules, for at least one isolate of CaMV,
with either several or just one discontinuity in the ( + ) strand (Maule & Thomas,
Structure a n d replication o f caulim ovirus genomes
219
1985) indicates that ( + ) strand synthesis can be either continuous of discontinuous, a
factor probably controlled by the availability of primers. Although the proportion of
molecules with genome-length ( + ) strands may be small, their existence will have to
be taken into account when deducing the mechanisms involved in reverse transcrip­
tion through an analysis of replication intermediates.
The replication complex
In vitro studies of CaMV DNA synthesis have been concerned firstly with the
identification of the DNA polymerase as a reverse transcriptase and secondly, but
equally, with attempts to understand the mechanisms involved, with reference
perhaps to what was already known about the amplification of retrovirus genomes.
With support from early autoradiographic evidence (Kamei et al. 1969; Favali et al.
1973) in favour of the inclusion body as the site of CaMV DNA synthesis, most
workers have opted for subcellular fractions enriched for inclusion bodies as the
starting material for the characterization of the DNA replicative activity. In
retrospect this was a sound decision since, although the inclusion body itself may not
be the primary structural unit in which or on which DNA replication occurs, it is
now generally accepted that the process does take place within its environs.
Pellet fractions obtained from low speed centrifugation of crude filtrates of tissue
extracts made in the presence of non-ionic detergents, contain large numbers of
inclusion bodies, together with nuclei from which they appear inseparable, but
relatively uncontaminated with other organelles. Further purification of inclusion
bodies (Mazzolini e ta l. 1985) and nuclei (Ansaei al. 1982; Guilboyle et al. 1983) has
been achieved, although preparations of either completely free from the other have
not been possible. Although the inclusion body preparations of Mazzolini et al.
(1985) contained some nuclear debris they did not contain any cellular DNA. These
low speed preparations incorporate labelled precursors into high molecular weight
products and more specifically into CaMV D N A (Pfeiffer & Hohn, 1983; Pfeiffer
et al. 1984; Guilfoyle et al. 1983; Modjtahedi et al. 1984; Mazzolini et al. 1985;
Marsh et al. 1985; Thomas et al. 1985) using endogenous templates, although the
proportion of the activity directed towards viral DNA synthesis may be low (e.g.
only 3 % of total DNA synthesis; Modjtahedi et al. 1984). Hypotonic leaching of the
replication complexes away from inclusion bodies followed by sucrose density
gradient separation yields peak fractions where CaMV DNA synthesis represents
8 0 -9 0 % of total DNA synthesis (Pfeiffer & Hohn, 1983; Pfeiffer et al. 1984) well
separated from CaMV-specific RNA synthesis. However, Marsh et al. (1985)
reported that this slowly sedimenting material did not have the properties expected
for a reverse transcribing replication complex.
An alternative strategy for the preparation of CaMV replication complexes gives
a clue as to their structural characteristics. We (Thomas et al. 1985) isolated
replication complexes from infected turnip tissue and protoplasts using an extraction
at pH 9, in the presence of Triton X -100, EG T A and high salt, and centrifugation
through a 6 0 % sucrose cushion at 250 000 £ , conditions sufficient to sediment par­
ticulate matter with a sedimentation coefficient in excess to 25S. These preparations,
220
R. Hull, S. N. Covey a n d A. J . M aule
without further purification, exhibited CaMV DNA synthesis as a larger proportion
(4 0 -5 0 %) of total synthesis than previously observed. The main difference between
this and other protocols was in the use of a high pH, high salt extraction, a procedure
which appears to disrupt inclusion bodies, since protein analysis of replication
complexes using antisera to CaMV O R F VI and O RF IV products, reveals very little
inclusion body protein but abundant coat protein (C. L . Harker, personal communi­
cation). T he association of replicative activity with viral coat protein in a particulate
form has also been noted by Marsh et al. (1985) who demonstrated the cosedimen­
tation of replication complexes from nuclear and microsomal lysates with intact
CaMV virions. Hence, a model for replication is suggested in which the virion or
provirion is implicated as the basic structural unit in which CaMV DN A synthesis
occurs. Several further pieces of evidence add support to this hypothesis. (1). The
detection in purified preparations of CaMV virions of CaMV DNA polymerase
activity associated with a protein of 76K , a size which is similar to that of the coding
capacity of the CaMV pol gene (Menissier et al. 1984). (2). The feature common to
both virion DNA and the products of in vitro replication in that both are released
from their nucleoprotein complexes by phenol only when predigested with protease
(Thom as et al. 1985; Marsh et al. 1985). (3 ). The observation that a class of single­
stranded CaMV DNA molecules, considered to be replication intermediates, can be
precipitated from preparations of replication complexes with antiserum to the 42K
coat protein (Marsh et al. 1985). (4). The belief that, for hepadnaviruses, the only
other retroid elements known to encapsidate the products of reverse transcription,
DNA synthesis and virion maturation are integral processes (see Mason et al. 1987).
The relationship between the specificity of the ‘cys’ motif on the gag or equivalent
gene products of retroviruses and CaMV and the tRNA used for priming ( —) strand
D N A synthesis (Covey, 1986) is interesting in this context, since it may indicate that
for CaMV there is a physical association between a coat protein subunit and the
initial priming event in DNA replication. Coordination of the processes of DNA
replication and encapsidation for caulimoviruses could account for the partial
insensitivity of the former to nucleases and other inhibitors noted above.
A M O D EL FOR THE REPLICATION COM PLEX
There is now enough information accruing for us to propose a model for the
replication complex. The data suggest that it resembles virus particles in many
respects. Further interpretation of the available information leads us to suggest that
there may be interactions between the inclusion body protein, the complete coat
protein gene product, the ( —) strand primer, the 35S RNA and eventually the viral
DNA.
T he viral coat protein is encoded by gene IV and is initially produced as a
precursor of 58K (Franck et al. 1980; Hahn & Shepherd, 1982). The capsid protein
is 42K which is further processed to products of 37 and 35K . The 58K precursor is
phosphorylated, the 42K protein derived from it is phosphorylated to a certain
Structure a n d replication o f caulim ovirus genomes
221
38 % R + K
M oM LV
p l5
PPl2
28% D + E
52% D
44% K
....................................................................... .
C aM V [
(ppii)
p42
A
j
(p4)
Fig. 2. Comparison of the structure of the gag gene product of a retrovirus, MoMLV and
CaMV. The proteins derived or thought to be derived from each main gene product are
listed below as p (protein) or pp (phosphoprotein) with the molecular weight in kDa.
Regions rich in aspartate and glutamate (D and E) or in basic amino acids (K = lysine,
R = arginine) are identified A = the ‘cys’ sequence CX2 CX9 C; H = hydrophobic region;
H = major capsid protein.
extent but there is little phosphorylation of the 37 and 35K proteins (Hahn &
Shepherd, 1980; Menissier-de Murcia et al. 1986). The latter polypeptides are
slightly.glycosylated (Hull & Shepherd, 1976; Duplessis & Smith, 1981). From a
comparison of the amino acid sequence of gene IV product with the amino acid
composition of CaMV coat protein, Franck et al. (1980) suggested that the
iV-terminal 100 or so amino acids (which are rich in aspartate and glutamate) and the
C-terminal 30 or so amino acids (which are also rich in aspartate) are removed in the
formation of the 42K protein. These regions of the 58K precursor protein are shown
diagramatically in Fig. 2 in which this gene is compared with the analogous gene, the
gag gene, of Moloney murine leukaemia virus (M oM LV). TheiV-terminal polypep­
tide of M oM LV (p l5) is very hydrophobic and interacts with the virion envelope. We
would suggest that the Ar-terminus of the CaMV 58K protein, the putative
phosphoprotein, p p ll interacts with the inclusion body protein via the aspartate­
glutamate rich region. The phosphorylated region of the 58K protein might also be
involved with the inclusion body protein or the nucleic acid. In MoMLV the
polypeptide (p p l2) between the hydrophobic p l5 and the main core protein, p30 is
phosphorylated and has been shown to be associated with the 35S RNA (Sen et al.
1976). But as Dickson et al. (1985) point out, the function of this protein is unclear
and it may also interact with other gag products. Hull & Covey (1986) have argued
that CaMV inclusion body protein is an analog of the retrovirus env protein. The
main capsid protein of both MoMLV (p30) and CaMV (p42) each contain a very
basic region which it has been suggested (Franck et al. 1980) is involved in nucleic
acid binding. Adjacent to, and C-terminal of, this in the CaMV 42K protein is the
‘cys’ m otif; this motif is in a similar relative position in MoMLV gag protein although
in the final processed product it is in an adjacent polypeptide (plO). The ‘cys’ motif is
considered to have Zn-mediated nucleic acid binding properties (Berg, 1986). As
noted above, Covey (1986) pointed out a loose correlation between the sequence
within the ‘cys’ motif and the tRN A used for priming ( —) strand DNA synthesis.
222
R. Hull, S. N. Covey a n d A. J . M aule
This raises the possibility that tRN A might interact with the ‘cys’ motif region and
that the upstream basic region interacts with other nucleic acid sequences.
From these considerations and those noted earlier in this chapter, we suggest the
model shown in Fig. 3. We assume, by analogy with retroviruses (see Varmus et al.
1982 for review), that gene V (th e pol gene) is expressed as a polyprotein with gene
IV (th e gag gene); a gag-pol polyprotein has recently been found in a hepadnavirus
(Will et al. 1986) and in a retrotransposon, the yeast T y element (Mellor et al. 1985;
Clare & Farabaugh, 1985; Kingsman et al. this volume). This assumption is not
essential for the model but it does help in positioning the polymerase. Also it is
difficult to reconcile the homology between the A'-terminal part of gene V product
with retrovirus protease domain if it is not expressed as a polyprotein. In the model
we suggest that, for the polyprotein, a linked association is set up between the
inclusion body protein and the A,T-terminal part of the 58K protein, the basic portion
of the 58K protein and the 35S RNA, the ‘cys’ motif and tRN Amet and between the
tR N A met and the primer binding site on the 35S RNA. This could then position the
RNA-dependent D N A polymerase subdomain of the gene V product and synthesis
of ( —) strand D N A could commence. There would have to be a mechanism that
ensured that a gene IV + V polyprotein, and not just a gene IV protein, associated
with the tRN A . If the putative p p ll was not cleaved from the 58K protein by the
protease domain on the gene V product then the gene IV produced on its own
without gene V could remain sequestered by inclusion body protein. Subsequent
steps in the assembly of the replication complex would include the release of gene V
from gene IV by the action of gene V protease, release of the 42K protein from the
association with inclusion body protein as the polymerase reached each molecule
attached to the 35S RNA and assembly of the 42K protein into virion-like structures.
It is realized that this model is highly speculative but it fits with current
observations and explains various features of the virus such as inclusion bodies and
the processing of the 58K coat protein precursor. However, if the model is
substantiated, there are various problems that will have to be explained. These
include the mechanisms of switching the interactions between the coat protein and
nucleic acid from single-stranded RNA to double-stranded DNA. The factors
involved in ‘maturing’ the virus particles to give the great stability found in purified
virions and, at the same time, removing most of the pol gene and the products of
RNase H digestion will have to be elucidated. Also to be explained is the occurrence
of the ‘unencapsidated’ replicative intermediates described earlier although these
could be associated with protein more tenuously.
Fig. 3. Diagram showing proposed model of the CaMV replication complex. In the top
left is an electron micrograph of a cytoplasmic viral inclusion body (bar marker - 0 - 5 fl).
In the top right is a diagrammatic representation of a portion of inclusion body with virus
particles embedded in it. Below is a diagram showing further details of the proposed
interactions between the inclusion body protein, the gene IV product, the gene V
product, the genomic RNA and the tRNA primer for minus-strand DNA synthesis. This
model is discussed in the text.
Structure and replication of caulhnovirus genomes
Inclusion body
protein^.-
Virus particle
Inclusion body
" protein
Gene IV
domains
Genomic
RNA
RNaseH
Polymera
Fig. 3.
Gelne V
domains
224
R. Hull, S. N. Covey a n d A. J . M aule
CaMV AND OTHER R EV E R SE T RA N SC RIBIN G ELEM EN TS
T he idea that CaMV replicated by reverse transcription first arose (Covey e t a l.
1983; Guilley e t a l. 1983; Hull & Covey, 1983; Pfeiffer & Hohn, 1983) because of the
structural similarities its genome exhibited with the genomes of hepatitis B viruses
(Summers & Mason, 1982) and retroviruses (see Weiss e t a l. 1982, 1985). These
included the presence of putative ( —) and ( + ) strand priming sites, a terminally
redundant genome-length RNA transcript (35S RNA) and the finding of CaMV ( —)
strand ‘strong-stop’ D N A (sa-D N A). In addition to these similarities there are
differences in detailed aspects of the replicative processes that presumably reflect an
evolutionary divergence and host adaptation in each element.
Both CaMV and the heapadnaviruses encapsidate in virions the DNA phase and
each exhibits a curious topology in that the DNA strands are discontinuous. One of
the early events in the replication cycles of both of these groups of viruses is thought
to be the generation of a supercoiled DNA transcription template which remains
extrachromosomal in the nucleus of infected cells. This template produces the
greater-than-genome-length RNA replication template as the next phase of the cycle.
In contrast, retroviruses package the RNA replication template phase in virions
and reverse transcription to produce DNA precedes the transcription phase in the
cycle. Moreover, the DNA which arises from reverse transcription of retrovirus
RNA is structurally complex in that considerable sequence rearrangement occurs to
produce a linear molecule with long terminal repeats (L T R s). This DNA provirus
becomes integrated into the host genome where it is transcribed to produce
messenger RNA and genomic RNA. In the transcription template of retroviruses
and in those of CaMV and hepadnaviruses an essential requirement is to generate
RNAs with terminal repeats. In retroviruses this is from a linear molecule by
transcription of the L T R s whilst in CaMV and hepadnaviruses the same sequence in
a circular (supercoiled) DNA is transcribed twice. In all cases this necessitates
ignoring transcription termination signals on the first pass but recognizing them on
the second. It is not understood how this is achieved. A further fundamental
difference in the replicative process of hepadnaviruses is that they are believed to
Fig. 4. Genome organization of cauliflower mosaic virus (CaM V), ground squirrel
hepatitis virus (G SH V ), Rous sarcoma virus (R SV ), Moloney murine leukemia virus
(M oM LV ), human T cell lymphotropic viruses II and I I I (H T L V II and H T L V III),
yeast T y element, Drosophila copia-Vike element 17-6 and Drosophila copia. Each is
shown as on the genome length RNA (----------), the terminal repeats being indicated by
( ■ ------ ). The subgenomic mRNA for the env gene or its equivalent is shown below the
genomic RN A; * indicates splice. The gag H, pol ■ and env B genes are highlighted,
genes I - V I I of CaMV, the b gene of G SH V , the src gene of R SV , the x gene of H T L V II
and the A and B genes of H T L V III are noted. The extents of the protease (pro), the
reverse transcription (R T ) and the integrase (int) domains of MoMLV and various
features and regions common to all or most genomes are shown: O = hydrophilic,
# = hydrophobic, p = phosphoprotein, o = nucleic acid binding domain, V = protease,
T = reverse transcriptase, <0> = integrase. References to the information in this Fig. are
given in the text. Reproduced from Hull & Covey 1986 with kind permission of the
Jou rn a l o f general Virology.
Structure and replication of caulimovirus genomes
225
prim e ( —) strand synthesis with a protein, while C aM V and retroviruses utilize a
host tR N A .
In addition to the sim ilarities in replicative m echanism s, these elem ents also share
certain basic com m on features in the organisation of th eir genes. T h is extends to a
large and diverse group of eukaryotic transposable elem ents called retrotransposons,
HTLV-II
n
„
IITLV-III
A
copia
^^gag'
m m p o l'
1/ 1 / \ env'
Dother
---------R N A
o hydrophi l i c
v prot ease
—* * — splice
• h y d r op h o b i c » r e v e r s e
n------ ■ repeat p phosphoprot ei n transcriptase
o n u c l e i c acid
° i ntegrase
binding domai n
Fig- 4.
226
R. Hull, S. N. Covey a n d A. J . M aule
the D N A phases of which bear striking similarity to integrated retroviral proviruses.
There is good evidence that at least two retrotransposons, the T y element of yeast
and the Drosophila copia element, undergo reverse transcription as part of the
transposition process (Garfinkel et al. 1985; Mellor et al. 19856; Shiba & Saigo,
1983).
As pointed out by Hull & Covey (1986) the genome organization of reverse
transcribing elements can best be compared in the RNA phase as shown in Fig. 4.
The archetypal retrovirus RNA genomes (of replication competent retroviruses)
comprises three gene domains: gag , pol and env. The gag gene encodes virion
structural polypeptides, pol the reverse transcriptase (and RNase H) and an
endonuclease activity thought to mediate provirus integration, and env encodes
virion envelope glycoproteins that are thought to confer host range specificity. A
similar organisation is exhibited by the other reverse transcribing elements but with
some modifications. For instance, some retrotransposons (e.g. T y element and
copia) have apparently lost (or have not acquired) the env gene which perhaps
reflects their properties as ‘non-infectious entities’. The AID S retrovirus, now
known as HIV (human immunodeficiency virus), has additional O R Fs that adapts it
to the infection of T 4 helper cells, the genetic organization of hepadnaviruses has
been ‘telescoped’ to produce a small genome with overlapping O R Fs, and the CaMV
genome has additional O RFs that presumably are required for its propagation in and
transmission between plant hosts. These variations aside, it would appear that
reverse transcribing elements share a common evolutionary origin and diverged at an
early stage although essential functions such as reverse transcription have been
conserved even at the amino acid sequence level. A similar suggestion has been made
by Mellor et al. (1986) and Kingsman et al. (this volume).
A further similarity between these elements is the rather unusual mechanism
adopted to express the^ag and pol genes in that it is linked to produce a polyprotein
subsequently processed into individual polypeptides. In the yeast T y element and
the retrovirus Rous sarcoma virus it has been demonstrated that frame-shifting
occurs between these two out of frame O RFs (Mellor et al. 1985a; Clare &
Farabaugh, 1985; Jacks & Varmus, 1985) and in CaMV there is evidence that
expression of at least three of its O RFs is linked (Sieg & Gronenborn, 1982; Dixon &
Hohn, 1984). It is interesting to speculate that such a frameshift, which on inspection
of Fig. 4 might be a common feature in reverse transcribing elements, might be
associated with the interaction of the genomic RNA and the upstream gag product
discussed earlier.
In conclusion, the reverse transcribing elements appear to constitute a unique
group in that although they have greatly diversified throughout the taxonomic groups
of eukaryotes, they have also retained many close similarities. It will be interesting to
see if they are wholly a eukaryotic phenomenon or whether their progenitors are to be
found amongst the prokaryotes as well.
Structure a n d replication o f caulim ovirus genomes
in
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