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Key words: reverse transcribing elements/genome organization
REVIEW ARTICLE
Genome Organization and Expression of Reverse Transcribing Elements:
Variations and a Theme
By R O G E R H U L L * AND S I M O N N. C O V E Y
Department o f Virus Research, John Innes Institute, Colney Lane, Norwich N R 4 7UH, U.K.
INTRODUCTION
Until recently, reverse transcription was considered to be the sole prerogative of retroviruses.
Over the last 4 or 5 years it has been recognized that members of two other virus groups, the
hepadnaviruses and the caulimoviruses, undergo reverse transcription during replication.
Furthermore, some vertebrate genetic elements, e.g. intracisternal A particle (IAP) genes (Ono
et al., 1985), VL30 genes (Hodgson et al., 1983) and L1Md (Loeb et al., 1986), and transposable
elements from other taxonomic groups [yeast Ty elements (Clare & Farabaugh, 1985; Hauber et
al., 1985), Drosophila copia (Mount & Rubin, 1985) and copia-like elements (Saigo et al., 1984),
Dictyostelium DIRS-1 element (Cappello et al., 1985), maize Bsl element (Johns et al., 1985), and
possibly maize Cinl element (Shepherd et al., 1984)] have been shown to possess structural
similarities to integrated retroviruses. The yeast Ty element transcript has recently been found
to be contained within virus-like particles which have reverse transcriptase activity (Garfinkel
et al., 1985 ; Mellor et al., 1985a). Details of the molecular biology of most of these viruses and
some of the elements have been reviewed recently (retroviruses: Weiss et al., 1982, 1985;
hepadnaviruses: Tiollais et al., 1985 ; caulimoviruses: Hohn et al., 1985; Covey & Hull, 1985;
Hull & Covey, 1985; all three groups of viruses: Mason et al., 1986; Ty and copia elements:
Boeke et al., 1985; Baltimore, 1985; Varmus, 1985). There is relatively little information on the
molecular biology of the other elements and we will not consider them further.
At first sight it might be thought that there were few similarities between these viruses and
transposable elements from such disparate sources. However, when their individual genetic
organization, expression and replication are compared it can be seen that they share certain
features which might be taken to indicate an evolutionary relationship. There are also some
differences which suggest that each element has adapted in particular ways to the genetic and
cellular environment in which it is propagated. It is the intention of this short review to explain
these similarities and differences.
Genetic organization
The genetic organization of reverse transcribing dements is best compared on the genomelength RNA transcript (Fig. 1) which is the template for reverse transcription. In each case, this
RNA (
) contains all the genetic information of the element; it also has a terminal
redundancy ( l - - l )
which can be from as little as 16 nucleotides in Rous sarcoma virus
(RSV) to about 300 nucleotides in human hepatitis B virus (Cattaneo et al., 1984). The terminal
repeat is considered to play an important r61e in the formation of negative-strand DNA from the
positive-strand RNA during reverse transcription. Retroviruses package this RNA in virions
and the equivalent transcripts of copia and Ty elements (Garfinkel et al., 1985; Mellor et al.,
1985a) have also been detected in virus-like particles. In contrast, caulimoviruses and
hepadnaviruses encapsidate the DNA phase in virions.
In most retroviruses, the 5' proximal open reading frame (ORF) is the gag (group-specific
antigen) gene ( . . . . a in Fig. 1), the protein products of which interact structurally with genomic
RNA to package it within the virions. Next comes the pol (polymerase) gene ( ~ - in Fig. 1)
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R. HULL AND S. N. COVEY
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Fig. 1. Genome organization of cauliflower mosaic virus (CaMV), ground squirrel hepatitis virus
(GSHV), Rous sarcoma virus (RSV), Moloney murine leukaemia virus (MoMLV), human T cell
lymphotropic viruses II and III (HTLV-II and HTLV-III), yeast Ty element, Drosophila copia-like
element 17.6 and Drosophila copia element. Each is shown on the genome-length RNA (
), the
terminal repeats being indicated by (11--11). The subgenomic mRNA for the gag gene or its
equivalent is shown below the genomic RNA; * indicates a splice. The gag ( ~ ) ,
pol (~m) and env
( ~ ) genes are highlighted. Genes I to VII of CaMV, the b gene of GSHV, the src gene of RSV, the X
gene of HTLV-II and the A and B genes of HTLV-III are noted. The extents of the protease (pro), the
reverse transcription (RT) and the integrase (int) domains of MoMLV and various features and regions
common to all or most genomes are shown: O, Hydrophilic; O, hydrophobic; p, phosphoprotein; <:~,
nucleic acid binding domain; V, protease; V, reverse transcriptase; O, integrase. References to the
information in this figure are given in the text.
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Review: Reverse transcribing elements
1753
encoding polypeptides with enzymic activities required in reverse transcription and integration.
As will be seen later, although the gag and pol genes of many retroviruses are in different reading
frames, they are expressed as a polyprotein. Thus, when discussing the function of domains
within them they are best considered as a unit. The retrovirus gag-pol polyprotein has the
following domains: NH2-structural polypeptides-protease-reverse transcriptase-endonuclease. In the reverse transcriptase domain of murine leukaemia virus and RSV, the RNase H
activity has been shown to be N-terminal relative to the DNA polymerase activity (Levin et al.,
1984; Grandgenett et al., 1985).
The hepadnavirus core antigen and caulimovirus coat protein (gene IV product) (Fig. 1) can
be considered to be analogous to gag as they are the gene products which interact with the
nucleic acid in the particle. The arrangement of gag and pol regions (the 'gag-pol' core) is thus
also apparent in hepadnaviruses, cauliflower mosaic virus (CaMV) and in retrotransposons
(Fig. 1). Organizational and amino acid sequence similarities between the retrovirus pol genes
and analogous genes of retrotransposons, CaMV and hepadnaviruses have already been noted
(see Toh et al., 1983; Varmus, 1985). These are highlighted in Fig. 1 where the protease (pro, V),
reverse transcriptase (RT) and endonuclease domains (int) of Moloney murine leukaemia virus
(MoMLV) are identified and the regions where amino acid sequence similarities exist between
the various elements are indicated. However, there are some differences in the arrangements of
the functional domains when retroviruses and the copia-like element 17.6 are compared with
copia and Ty 912 (Mount & Rubin, 1985). Thus, in copia and Ty, the reverse transcriptase (V)
and integrase (O) domains of the pol region are apparently inverted relative to the other elements
(Fig. 1).
As noted above, a general feature of reverse transcribing elements is that polypeptides derived
from the gag ORF interact with genomic nucleic acid to give virus or virus-like particles. This
similarity extends even to regions within the gag ORF. The protein derived from the C-terminal
region of the gag product is rich in basic amino acids ( ~ in Fig. 1) and in retroviruses is known to
bind to the genomic RNA. The amino acid sequence CX2CXgC, highly conserved in retrovirus
nucleic acid binding proteins (Henderson et al., 1981 ; Copeland et al., 1984), is found in ORF I
of copia (Mount & Rubin, 1985) and the coat protein of CaMV (Covey, 1986; M. Pietrzak, J.
Fuetterer & T. Hohn, personal communication), although it is not found in the Ty element, the
eopia-like 17.6 element or in hepadnavirus core antigen. For many retroviruses, one of the
proteins derived from the N-terminal portion of the gag polyprotein is phosphorylated (p in
Fig. 1) although the phosphoprotein is apparently absent from the gag product of human T cell
lymphotropic virus (HTLV-III)now thought to be a lentivirus (see Rabson & Martin, 1985). The
gag proteins of hepadnaviruses, CaMV and the Ty element are also phosphorylated (p in Fig. 1).
From the data of Franck et al. (1980) and Hahn & Shepherd (1980) it can be deduced that the
phosphorylated region in the CaMV coat protein precursor, which undergoes processing, is in
the N-terminal half and so is in an analogous position to the phosphoprotein P 10-P 17 of various
retroviruses. The phosphorylated domains of the gag proteins of hepadnaviruses and Ty
elements have not yet been identified. The N-terminal part of CaMV coat protein is hydrophilic
(© in Fig. 1) in contrast with the hydrophobic N-termini of some retrovirus gag polyproteins
(MoMLV, RSV) and of the equivalent gene product (core antigen) of hepadnaviruses (Q in Fig.
1). This probably reflects the fact that retroviruses and hepadnaviruses assemble in association
with membranes whereas CaMV assembles in viral inclusion bodies.
For copia and Ty elements, all of the coding capacity is taken up by the gag-pol core whilst the
other viruses and retrotransposons have additional coding regions. Those viruses that infect
vertebrate cells (the retroviruses and hepadnaviruses) have an env gene or surface antigen (,,, ,,
in Fig. 1). This coding region forms one or more glycoprotein(s) which are exposed on the virus
capsid surface and interact with the host cell receptors. The third ORF in the copia-like element
17.6 has been suggested to be analogous to the retrovirus env gene (Saigo et al., 1984). In CaMV,
the ORF (gene VI) immediately downstream from the pol gene (gene V) codes for a major
protein component of inclusion bodies. These are cytoplasmic proteinaceous structures which
contain most of the virus particles and are considered to be the sites of reverse transcription and
virion assembly (see Maule, 1985; Hull & Covey, 1985). Thus, this gene product envelopes the
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R. HULL
AND
S. N . C O V E Y
CaMV replication function as do the env gene products of retroviruses and hepadnaviruses;
however, in caulimoviruses it forms a type of syncytium in that the inclusion body protein forms
an aggregate containing many virus genomes.
CaMV has four additional ORFs (genes VII, I, II and III) 5' to the gag-pol core (Fig. 1). We
have recently suggested (Hull & Covey, 1985) that ORF I is involved in cell-to-cell spread of
CaMV within susceptible plants. The ORF II product facilitates aphid transmission of the virus
from plant to plant (Armour et al., 1983 ; Woolston et al., 1983 ; Givord et al., 1984). No function
has yet been ascribed to ORFs III and VII although ORF III is expressed as a protein in plants
and is indispensable for virus infection (Xiong et al., 1984). ORF VII is dispensable (Dixon &
Hohn, 1984) and it is questionable whether it produces a functional protein.
Gene expression
Common features of the genome organization and replicative mechanism are reflected in the
strategies of gene expression adopted by reverse transcribing elements. The DNA phase is the
starting point for gene expression, although this transcription template generates RNA
molecules that serve two functions: replication and protein synthesis. There are differences in
the location and structure of transcription templates for each group of elements. For all elements
that have been studied, transcription occurs in the nucleus and is directed by the RNA
polymerase that normally transcribes host protein-encoding genes. It is not surprising, therefore,
that nucleotide sequences involved in the control of transcription initiation and termination of
host genes are located in the appropriate positions within the DNA phase of reverse transcribing
elements.
In most retroviruses and retrotransposons, the transcribed DNA assumes a linear
configuration as it is integrated in the host genome. An exception to this amongst the
retroviruses is visna virus, a member of the lentivirus group, the majority of the complementary
DNA forms of which are linear molecules that remain extrachromosomal in tissue culture cells
(Haase et al., 1982; Harris et al., 1984). In contrast, the transcription template of hepadnaviruses
is considered to be an extrachromosomal supercoiled DNA (see Mason et al., 1986 for
discussion). Integrated forms of the DNA of some hepadnaviruses have been found (see Mason
et al., 1986) although these are not thought to play an essential r61e in the virus multiplication
cycle. CaMV DNA also becomes a supercoiled component of an episomal minichromosome
(Olszewski et al., 1982) which is the template for transcription. In addition, a complex
population of subgenomic forms of CaMV supercoiled DNA have been detected in infected
plants (Olszewski & Guilfoyle, 1983; Rollo & Covey, 1985) but their function remains to be
determined.
Following transcription, mRNAs move to the cytoplasm for translation. It has been known
for some time that, in retroviruses, translation of the gag and pol regions is linked. The gag
polypeptides that constitute the virion core are generated via two routes (see Varmus, 1985).
First, an RNA, thought to be similar in structure to genomic RNA, is translated to produce agag
precursor polyprotein. Additionally, a longer gag-pol polyprotein is generated at a level 1 to 10 ~o
that of the gag protein alone. Polyprotein processing is thought to be directed by the protease
domain (XZ in Fig. 1) of the gag-pol polyprotein discussed above. The mechanism by which the
gag-pol polyprotein is generated has been the subject of speculation since the two ORFs are
separated by a stop codon or by being in different reading frames (Fig. 1). For some retroviruses
like MoMLV, the two in-frame genes are separated by an amber termination codon which is
thought to be suppressed, resulting in gag-pol synthesis (Yoshinaka et al., 1985). For many other
retroviruses, e.g. RSV and HTLV-III, the gag andpol ORFs overlap (in a different frame) and in
human T cell leukaemia virus type II (HTLV-II), the gag, protease and pol ORFs overlap in
three different reading frames (see Fig. 1); spliced RNAs containing two or three frames fused
have never been found. However, recent studies of the yeast Ty retrotransposon have provided
evidence that a frameshift mechanism operates at the overlap of the gag andpol ORFs such that
a gag-pol polyprotein is synthesized from the two out-of-frame genes (Mellor et al., 1985 b; Clare
& Farabaugh, 1985). Moreover, Jacks & Varmus (1985) have generated an RNA species in vitro
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by transcription of an SP6 construct that contained the out-of-frame gag and pol ORFs of RSV
and showed that, following in vitro translation in a reticulocyte lysate, a fusion protein was
produced. This strongly supports the view that frameshifting occurs more widely amongst
reverse transcribing elements in the synthesis of the gag-pol polyprotein.
In hepadnaviruses, the first two ORFs in the genome-length transcript, the core antigen ORF
( ~ in Fig. 1) and the polymerase ORF (-..~ in Fig. 1), analogous togag andpol, overlap in a
manner reminiscent of retroviruses. This perhaps implies that frame shifting might also
generate the appropriate polyprotein since spliced RNAs of hepadnaviruses have not been
observed. However, a fusion protein has not been detected in infected cells either and the
conserved protease domain ( V in Fig. 1), found in the gag-pol region of other reverse
transcribing elements, which might function in processing a polyprotein, is absent from
hepadnaviruses (Varmus, 1985).
For CaMV, the expression of the gag and pol genes (ORFs IV and V) is also not well
understood. Unlike most other reverse transcribing elements, these two ORFs are not located
close to the 5' end of the major genome length transcript but instead have four ORFs preceding
them. However, there is more than just a suggestion that translation of the first four CaMV
ORFs (VII, I, II and III; see Fig. 1) is linked. The evidence comes from studies of the effects,
upon viral DNA infectivity, of insertions and deletions in two non-essential ORFs (VII and II;
see Fig. 1) which have shown that insertion of an AUG codon leads to loss of infectivity if it is
not followed by an in-frame termination codon. Furthermore, initiation and termination codons
of adjacent ORFs must be located close to one another for infectivity to be retained (Sieg &
Gronenborn, 1982; Dixon & Hohn, 1984). These studies have suggested that a 'relay race'
mechanism of translation occurs on the CaMV genome-length transcript in which ribosome
small subunits do not completely disengage at the end of a given ORF and protein synthesis is reinitiated internally in the RNA (Sieg & Gronenborn, 1982). Because ORFs I to III tandemly
overlap one another slightly (see Fig. 1), this makes possible a frameshifting mechanism similar
to that demonstrated for Ty and RSV. However, the appropriate polyprotein that should be
generated has not been observed in CaMV-infected cells. Moreover, a frameshifting expression
mechanism would not explain why each ORF has a Y-proximal AUG initiation codon adjacent
to the termination codon of the preceding ORF.
Deletion and insertion analysis has not been possible for the CaMV gag andpol ORFs (IV and
V) because both are essential genes and so it remains an open question as to whether
frameshifting, along the lines proposed for retrovirus and yeast Ty gag-pol ORFs, generates the
appropriate CaMV polyprotein(s). The existence ofa CaMV polyprotein is suggested, however,
because its pol gene (ORF V) contains the protease domain (V) conserved in retroviruses and
retrotransposons (Fig. 1). The story is still far from clear though for CaMV, since little is yet
known concerning the fine structure of mRNAs that express ORFs VII to V, a situation thatis
further complicated by the suggested existence of a separate subgenomic mRNA for the pol
ORF possibly required for specific gene amplification (Plant et al., 1985), and the inference from
the observations of Hirochika et al. (1985) that other ORFs might be fused by RNA splicing.
The env ORF of reverse transcribing elements (, • • ~in Fig. 1) in general lies downstream of
the gag-pol core. In some elements, e.g. CaMV and HTLV-III, the poi and- env ORFs are
separated. In others, e.g. RSV and MoMLV, env slightly overlaps the end of pol whilst in
hepadnaviruses, env (the surface antigen gene;Ezzn) completely overlaps pol. These variations
aside, the env ORF in each type of element for which data are available is expressed via a
subgenomic mRNA that is 3' co-terminal with the genome length RNA (see Fig. 1), The
retrovirus env mRNA has the 5' leader of the genomic RNA spliced onto it (--* *-- in Fig. 1)
whilst the equivalent CaMV mRNA is transcribed under the direction of its own promoter (see
Covey, 1985). The means by which the subgenomic mRNA of the hepadnavirus ground squirrel
hepatitis virus (GSHV) (Enders et al., 1985) is generated is not yet known although, like the
CaMV transcript, it is not spliced. Similarly, a separate transcript for the copia-like
retrotransposon 17.6 env gene has not been observed.
Thus, allowing for the limitations of information available noted above, it would appear that
for the viruses and elements which use reverse transcription in their replication, the gag-pol core
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R. H U L L AND S. N. COVEY
is one functional unit and the env gene is another; the transcripts of these two units appear to be
3' co-terminal.
DISCUSSION
We have drawn attention above to similarities and differences in the genetic organization of
reverse transcribing viruses and elements. We would like to propose that these genomes have a
gag-pol core to which is added cassettes of genes necessary for their propagation within their
own particular host system. For viruses that infect vertebrates, the env gene product is required
for cell-to-cell spread and it also encloses the reverse transcription machinery, a function
possibly performed by the gene VI product of CaMV and that of the third ORF in the copia-like
element 17.6. In CaMV, there are cassettes comprising the gene required for transmission from
plant to plant (gene II) and the putative gene for cell-to-cell spread (gene I; see Fig. 1). The Ty
and copia elements do not appear to need host-adapting cassettes of genes. Since the
reproduction of yeast is either by binary fusion or cell fusion, and because yeast cells have a thick
cell wall, there would appear to be no requirement for cell-to-cell spread factors to transmit Ty
sequences. There is no such limitation to the cell-to-cell spread of viruses in Drosophila although
it is not known ifcopia particles spread in this manner. Even if they do not, both Ty and copia can
be considered as being transmitted from cell to cell during division as integrated elements in the
chromosomal DNA or as episomal particles. Fig. 1 shows that some of the viruses also carry
other genes, e.g. A and B in HTLV-III (see Rabson & Martin, 1985) and b in GSHV (Seeger et
al., 1984); the full significance of these genes to the host adaptation of the virus is not yet
understood although it has been suggested that the gene product of the human T cell leukaemia
viruses types I and II ORF X has a trans-acting effect upon transcription and is involved in the
cell specificity of transformation (Sodroski et al., 1984). Very recently, a trans-activating gene,
called tat-Ill, has been found for HTLV-III. tat-Ill is not shown in Fig. 1 as it comprises three
exons, one non-coding region from the 5' long terminal repeat, one coding region from between
the A and env genes (Fig. 1) and one within the env gene in a different reading frame (Arya et al.,
1985; Sodroski et al., 1985). The product of the tat-III gene is an absolute requirement for virus
expression and replication in human T lymphocytes (Fisher et al., 1986).
How these cassettes of genes have been collected together and where they come from are, as
yet, unanswered questions. In his protovirus hypothesis, Temin (1974) suggested that
retroviruses evolved from normal cellular components. An extension of this hypothesis is that
the formation of the gag-pol core represents a single evolutionary event. This core has spread
across a wide range of taxonomic groups acquiring other cassettes of genes to enable adaptation
to specific hosts. It will be interesting to see if cellular relatives of the pol gene of reverse
transcribing elements turn out to be on their own, or associated with progenitor gag sequences.
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