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Journal of Virology, October 2000, p. 9776-9785, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Alphavirus RNA Genome Repair and Evolution: Molecular
Characterization of Infectious Sindbis Virus Isolates Lacking a
Known Conserved Motif at the 3' End of the Genome
Jyothi
George and
Ramaswamy
Raju*
Department of Microbiology, School of
Medicine, Meharry Medical College, Nashville, Tennessee 37208
Received 22 May 2000/Accepted 25 July 2000
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ABSTRACT |
The 3' nontranslated region of the genomes of Sindbis virus (SIN)
and other alphaviruses carries several repeat sequence elements (RSEs)
as well as a 19-nucleotide (nt) conserved sequence element (3'CSE). The
3'CSE and the adjoining poly(A) tail of the SIN genome are thought to
act as viral promoters for negative-sense RNA synthesis and genome
replication. Eight different SIN isolates that carry altered 3'CSEs
were studied in detail to evaluate the role of the 3'CSE in genome
replication. The salient findings of this study as it applies to SIN
infection of BHK cells are as follows: i) the classical 19-nt 3'CSE of
the SIN genome is not essential for genome replication, long-term
stability, or packaging; ii) compensatory amino acid or nucleotide
changes within the SIN genomes are not required to counteract base
changes in the 3' terminal motifs of the SIN genome; iii) the 5' 1-kb
regions of all SIN genomes, regardless of the differences in 3'
terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in
the SIN genomes carrying defective 3'CSE, these are not essential for
genome viability or function; and v) the newly added AU-rich motifs are
composed predominantly of RSEs. These findings are consistent with the idea that the 3' terminal AU-rich motifs of the SIN genomes do not bind
directly to the viral polymerase and that cellular proteins with broad
AU-rich binding specificity may mediate this interaction. In addition
to the classical 3'CSE, other RNA motifs located elsewhere in the SIN
genome must play a major role in template selection by the SIN RNA polymerase.
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TEXT |
Viruses carrying RNA genomes cause
devastating human illnesses, such as AIDS, rabies, poliomyelitis,
hepatitis, and hemorrhagic fevers. There is little doubt that these
viruses evolve rapidly in response to environmental and genetic
pressures (25, 57). For example, hundreds of different
genotypes of recombinant human immunodeficiency virus (HIV) genomes
contribute to the growing AIDS epidemic (7). Viruses such as
influenza undergo continuous genetic changes and escape host immune
mechanisms (63). The polymerases and cellular factors that
act upon RNA genomes are central to the survival and evolution of RNA
viruses. Despite our expanding knowledge of the biology of RNA viruses,
understanding the anatomy and biochemistry of the enzymes and factors
that copy and modify the RNA genomes continues to be challenging. RNA
genomes in general are believed to carry cis-acting RNA
motifs that regulate RNA synthesis and genome amplification. Many RNA
genomes, including those of all retroviruses, carry these regulatory
RNA motifs at their genome termini (12, 57). However,
results obtained from some animal and plant RNA viral systems suggest
the occurrence of internal cis-acting motifs (2, 16,
19, 38, 40, 41, 50). Since the cis-acting RNA motifs
presumably interact with viral and cellular factors, the evolution
of these RNA motifs is thought to be constrained. In
fact, the sequence and/or structure of these RNA motifs are
well conserved among members of a given RNA virus family and even among
members of closely related families. Mutational changes in these
conserved sequence elements (CSEs) could have lethal effects on virus
replication unless compensatory genetic changes occur within the
protein factors that bind to these CSEs.
Alphaviruses are mosquito-transmitted animal and human RNA viruses and
are the predominant component of the Togaviridae family (29). Alphaviruses cause fever, arthritis, skin rashes, and encephalitis in humans and livestock. Alphaviruses are extensively distributed in both the old and new worlds and are responsible for
periodic outbreaks of human illnesses (29, 59). All
alphaviruses carry a 12-kb positive-sense RNA that encodes a very
similar set of polymerase and structural proteins from two open reading
frames (53, 59). Alphavirus genomes resemble eucaryotic
mRNAs in that they possess 5' cap structures and 3' poly(A) tails (Fig. 1A). In addition, the 5' and 3' proximal
sequences of alphavirus genomes carry differing lengths of
nontranslatable regions (NTRs) that are believed to carry
cis-acting motifs which regulate viral RNA synthesis. At
least four CSEs, or RNA motifs, are found in all alphaviruses (Fig. 1A)
(59). These CSEs are thought to bind to viral and/or
cellular proteins and regulate viral RNA synthesis. Genetically
engineered DNA copies of alphavirus RNA genomes have been used
extensively to study the roles of RNA motifs and proteins in virus
replication and pathogenesis (20, 23, 29, 33, 39, 46, 47, 51,
65). The use of alphavirus vectors in nucleic acid-based gene and
vaccine delivery applications has stimulated much interest in the
biology of alphavirus vectors (17, 18, 26, 37, 54).
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FIG. 1.
(A) Organization of the SIN genome. NS, coding region
for nonstructural proteins; S, coding region for structural proteins;
5'CSE-1, the 44-nt 5' conserved sequence element at the 5' end;
5'CSE-2, the 51-nt 5' conserved sequence element located within the 5'
translatable region; JN-CSE, the 21-nt-long conserved sequence element
located at the junction of the NS and S coding regions that serves as a
promoter for subgenomic RNA synthesis; 3'NTR, the 0.3-kb 3'
nontranslated region that carries several repeat sequence elements;
3'CSE, the 19-nt conserved sequence element located at the 3' end
adjoining the poly(A) tail. The drawing is not according to scale. (B)
Sequences of the 3'NTR domain of eight SIN isolates. These eight SIN
isolates, which were recovered from individual plaques (46),
were used to infect BHK cells to generate virus stocks. The
virus-specific RNAs obtained from infected BHK cells were reverse
transcribed with 18TSac and amplified by PCR using the primers T11200
and 18TSac as previously described (46). The single
species of PCR product obtained for each of the isolates was purified
and sequenced. Each isolate is identified on the left of each sequence.
The sequence 1101-RR corresponds to the SIN derived from the parental
clone Toto 1101 (39, 47, 51). The control isolate S3-7 was
generated from the SIN clone Tapa (51). Since the Tapa
plasmid was derived from Toto 1101, it was expected to carry identical
protein-coding regions. The Tapa plasmid carries an intact 3'NTR
and an ApaI restriction site at the 3' end of the S-coding
region. The location of the ApaI site (gggccc) and the
stop codon (tga) are shown. Notations used: lowercase
letters, original sequence of the template used for virus production;
underline, bases corresponding to the 3'CSE; uppercase letters,
bases inserted during genome repair in vivo; dots, base deletions;
hyphens, base identity; back-slashes, discontinuity in sequence used
for drawing purposes.
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Sindbis virus (SIN) is one of the best-studied alphaviruses at the
molecular level (59). The SIN genome carries a 0.3-kb 3'NTR
and a poly(A) tail, whereas the sizes of the 3'NTRs of other alphavirus
genomes range from 80 to 610 bases (45). Despite this wide
size difference, all 27 known alphavirus genomes carry a very highly
conserved 19-nucleotide (nt) motif at the 3' end of the genome just
upstream of the poly(A) tail (45). In addition, several
repeat sequence motifs (RSEs) and strong secondary structures also
occur in the 3'NTR in alphaviruses (32, 43, 45). The 3'
19-nt CSE, along with the poly(A) tail, has long been believed to
function as the RNA promoter for the initiation of negative-sense RNA
synthesis from the positive-sense genome (32, 35, 36). Contrary to these expectations, we recently showed that SIN genomes lacking a part or all of the 19-nt 3'CSE were able to initiate replication and produce infectious virus (24, 46).
Surprisingly, analysis of the 3'NTR sequences of the progeny virus
genomes demonstrated the presence of either the parental mutant 3'CSE
or differing lengths of new AU-rich motifs at the initial site of
mutation (46). In essence, upon transfection into BHK cells,
a given species of in vitro-synthesized SIN RNA carrying a defined
deletion in its 3'CSE gave rise to a population of viruses that
differed in the size of the AU-rich 3' terminal region of their genomes (46). It was not clear how the viral polymerase was able to recognize the progeny genomes carrying AU-rich motifs of heterogeneous size at their 3' termini. In this report, we present further
information on these novel SIN genomes, including the stability of the
altered 3'NTRs, growth properties, RNA synthetic abilities, and genome organization and sequences of representative SIN isolates.
Virus isolates and their growth properties.
Previously we
reported the isolation of a library of infectious SIN mutants that
carried significant deletions and/or additions in the 19-nt CSE of the
3'NTR of these RNA genomes (46). The library was obtained by
transfecting BHK cells with the in vitro-transcribed SIN RNAs carrying
deletions within the 3'CSE. The culture supernatants recovered from the
BHK cells were subjected to a single round of plaque purification, and
the individual plaque suspensions were stored frozen. These plaque
suspensions were assigned the passage zero (p-0) designation: they had
a virus titer of 103 to 104 PFU in each
suspension. Representative plaque suspensions were used to infect BHK
cells at a multiplicity of infection of 0.1 PFU/cell, and virus stocks
were prepared. These virus stocks were designated passage 1 (p-1). The
virus titers of p-1 stocks of all isolates were in the range of
107 PFU/ml. To understand the biological properties and
mechanism of the origin of these viruses, we chose to analyze in depth
eight isolates, whose 3'NTR sequences are shown in Fig. 1B. These
isolates carried different lengths of the 3'NTRs due to the addition of different sizes of AU-rich motifs adjacent to the poly(A) tail. The
plaque sizes of these isolates were similar, except that the plaque
size of the isolate S3-4 was ca. 20% smaller than that of other
isolates (Fig. 2). To test the stability
of these variant 3'NTRs and to evaluate the growth properties of these
virus isolates, duplicate BHK cell cultures were infected with 3 to 5 PFU of the different SIN isolates/cell and adsorbed at 30°C for
1 h, the inoculum was removed, and 1 ml of fresh medium was added.
The cytopathic effect (CPE) was monitored every 2 h for the first 10 h of infection. The final CPE was recorded around 24 to 26 h postinfection (p.i.), the culture supernatant was recovered, and the
titer of virus was determined. The virus stocks recovered from the p-1
were used to infect fresh BHK cultures, and the extent of CPE and virus
titer were determined. Eighteen such passages were carried out, and CPE
and virus titer were determined. As shown in Table
1, the overall difference in virus titer
between the isolates and passages varied only up to three- to fourfold. All the isolates except S3-20 produced significant CPE around 7 to
8 h p.i. All the isolates produced extensive CPE and cell detachment of BHK cells when observed around 24 to 26 h p.i. Since the total amounts of virus released from all the isolates did not
differ significantly, the kinetics of virus release was determined using the p-1 virus isolates. As shown in Fig.
3, the isolate S3-20, which carried a
13-nt deletion in the 3'CSE, showed a significant delay in release of
virus particles early during virus infection, but around 9 to 10 h
p.i., virus release paralleled that of the other isolates. These
results demonstrated that the genomes of the SIN isolates carrying
diverse 3'NTR sequences with drastically altered 3'CSE retain a high
level of infectivity and release comparable levels of virus particles
from BHK cells for all the 18 passages tested.
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FIG. 2.
Plaque sizes of SIN isolates. Confluent Vero cultures
were infected with p-1 stocks of the eight virus isolates, overlaid
with agar, and incubated at 37°C. Around 50 to 54 h p.i.,
plaques were fixed with paraformaldehyde, stained with crystal violet,
and photographed. Note that the plaques of isolates S3-7 and S3-23 were
larger, whereas those of S3-10 and S3-20 were smaller. The plaques of
isolate S3-10 were clear, but the plaques of all other isolates were
somewhat diffused.
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FIG. 3.
Time course of virus release. Duplicate BHK
cell cultures were infected with 6 PFU of p-3 SIN isolates/cell and
incubated at 37°C for 1 h, the inoculum was removed, and the
cells were replenished with 1 ml of medium containing 2% fetal bovine
serum. At 3, 5, 7, 9, and 11 h p.i., culture supernatants were
completely removed, and 1 ml of fresh medium was added to the cells.
The amount of infectious virus found in the recovered medium was
determined by plaque assay. Each value represents the average of two
experiments. In the two experiments, the isolate S3-20 showed very
similar growth kinetics.
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The level of virus-specific RNA synthesized in the infected cells
depends heavily on the nature of its gene regulatory elements.
Since
the 3'CSE and the 3'NTR are thought to play major roles
in polymerase
recognition, RNA synthesis, and genome translation,
it was thought
likely that the level of virus-specific RNA synthesized
from test
isolates would be different. To investigate this possibility,
BHK cells
were infected with each of the p-1 virus isolates at
a multiplicity of
infection (MOI) of 4 to 6 PFU/cell, and the
level of virus-specific RNA
synthesized between 5 and 9 h p.i.
was determined by metabolic
labeling. As shown in Fig.
4, the
steady-state levels of genomic RNA varied up to 40%, and the ratio
of
genomic RNA levels to subgenomic RNA levels ranged from 1.3
to 1.5. Since these differences are small, the terminal sequences
of the 3'NTRs
as found in the eight virus isolates appear to have
had little effect
on the steady-state levels of virus-specific
RNAs. Although the growth
kinetics of the isolate S3-20 was significantly
impeded (Fig.
3), the
virus-specific RNA levels were unaffected.
Similar virus-specific RNA
levels were found in BHK cells infected
with p-18 virus isolates (data
not shown).
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FIG. 4.
Levels of virus-specific RNA expressed by SIN
isolates. BHK cell cultures were infected with 6 PFU of each virus
isolate/cell and labeled with [3H]uridine between 5 and
9 h p.i. as previously described (46, 47). In brief,
infected cells were replenished with 0.6 ml of minimal essential medium
containing 3 µg of dactinomycin per ml at 6 h p.i. Twenty
minutes later, 50 µCi of [3H]uridine (Dupont-NEN) was
added to each plate, and the infection was continued at 37°C for an
additional 3 h. At the end of the labeling period, cells were
harvested in phosphate-buffered saline and disrupted with 1% NP-40,
and the cytoplasmic supernatants were recovered by centrifugation.
Cytoplasmic RNA was purified by phenol-chloroform extraction and
precipitation with two volumes of ethanol (46, 47). The
amount of RNA recovered was determined by UV spectrophotometry. Six
micrograms of the isolated RNA was denatured with glyoxal, analyzed on
a 1.25% agarose gel, and then fluorographed as described previously
(46, 47). The radioactivity corresponding to each of the
bands was recovered, solubilized in a biodegradable solvent (BCS;
Amersham) and quantitated. For comparison, the intensity of the RNA
bands was also determined by densitometry using the Gel-doc 2000 apparatus and Quantity One image analysis software (Bio-Rad). The top
of each lane indicates the identity of the isolate. UI, uninfected; g,
genomic RNA, 11.7 kb; sg, subgenomic RNA, 4.2 kb; g/sg,
ratio of genomic RNA to subgenomic RNA. The values were
determined from two experiments. RNA samples derived from each
experiment were loaded twice on the same gel for densitometry and
determination of radioactivity.
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Sequence changes in genomes of different SIN isolates.
Results
reported above indicated that the majority of the p-1 virus isolates
carrying different 3' terminal sequences grew well and synthesized
comparable levels of virus-specific RNAs. Although these results were
unexpected, we thought that additional compensatory sequence changes
could have occurred elsewhere in the genome. Therefore, we sequenced
the entire genome of all eight virus isolates. In brief, cytoplasmic
RNA isolated from infected BHK cells was reverse transcribed with an
oligo(dT) primer and amplified by PCR with 12 sets of primer pairs to
generate overlapping DNA fragments of ca. 1 kb. These DNA fragments
were purified to remove the oligonucleotides and sequenced using two to
four primers specific to each of the DNA fragments. To determine the
precise sequence of the 5'-terminal region of the genomes, the
first-strand cDNA products corresponding to the 5' 1-kb region were
tailed with dC and were amplified by PCR and sequenced. The sequence data obtained from this analysis were compared with the published sequence of a SIN strain known as HRsp (58). As shown in
Table 2, several base changes
between the genome of the HRsp strain and that of the SIN
isolates reported here were demonstrated. Since all the SIN
isolates used in this study were derived from the parental SIN cDNA
clone Toto 1101 or its derivatives (39, 51), it was
important to establish whether or not the corresponding base changes
could be found in the Toto 1101 DNA. It was also possible that some of
these base changes could have occurred during the in vitro synthesis of
RNA from these DNA templates. To explore these possibilities, two
parental DNA templates, namely Toto 1101 and T3'15 (46),
were used as controls. These two plasmids were transcribed in vitro as
described previously (46) and treated with DNase I to remove
template DNA, and the resulting RNA preparations were used as templates
for reverse transcription and amplification by PCR. PCR products were
then sequenced to establish the identity of the bases reported in Table
2. Finally, the nucleotide positions 353, 2992, 2579, and 5702 of Toto
1101 plasmid DNA were sequenced using Sequenase as a test to rule out
possible artifacts of automated sequencing.
Table
2 summarizes the positions and base changes in the protein coding
sequences of isolates that were sequenced. Overlapping
DNA fragments
corresponding to the entire genomes of all eight
isolates (S3-7 to
S3-23) were sequenced and assembled using the
DNA assembly software
Sequencher (Gene Codes Corp). In brief,
5 µg of the infected cell RNA
from each of the eight SIN isolates
was reverse transcribed with the
primer 18T Sac, and the first-strand
cDNA was purified by digestion
with RNase H and RNase T1 enzymes
following the manufacturer's
protocol for the 5' RACE kit (GIBCO/BRL).
The purified products were
used as a template for PCR amplification.
The primer pairs used to
amplify the approximately 1-kb region
of the SIN genome from the 5' end
to the 3' end were the following:
T32, T1050
; T950, T2050
; T1950,
T3100
; T3050, T4150
; T4050,
T5250
; T5200, T6350
; T6300,
T7350
; T7300, T8300
; T8200, T9050
;
T9000, T10050
; T10000,
T11000
; and T10950, 18TSac
(Table
3).
The single species of DNA product obtained from these PCRs was
purified
by a DNA purification kit (Mo Bio Laboratories) and sequenced
using the
Bigdye terminator kit (ABI). In addition to the primers
used in the
individual PCRs, additional oligonucleotides were
used in sequencing to
verify the overlapping regions of the PCR
products. To determine the
exact 5' terminal sequence of the SIN
genomes, the infected cell RNA
was reverse transcribed with a
negative-sense primer, T1050
, and the
cDNA was purified using
the 5' RACE kit (GIBCO/BRL), tailed with dCTP,
and amplified by
PCR using the abridged anchor primer provided in the
kit and the
primer T1050
(Table
3). The PCR products obtained from
these
reactions were purified and sequenced using the primers T200
,
T500
, and T1050
(Table
3). The mismatched bases between the
published genome of the HRsp strain of SIN (
58) and those of
isolates reported in this study are shown. At least nine base
changes
were found within the protein-coding sequences of the
Toto 1101-RR
strain compared to the HRsp strain. The nucleotide
sequences of the
protein-coding region of isolates S3-7, S3-8,
S3-11, S3-20, S3-4,
S3-23, and Toto 1101-RR were identical (Table
2). Isolate S3-10 carried
a C to G (Leu to Val) change at position
2133 in the nsp2-coding
region. Isolate S3-9 carried a C to U
change at position 4874, with no
amino acid changes. As shown
in Fig.
1B, all isolates carried an
identical 3'NTR, except for
the terminal AU-rich motifs. S3-23 carried
a C residue instead
of U at position
226. As shown in Fig.
5, no base changes were
found within the
5'NTR domains of any of these isolates compared
to that of the
wild-type isolate S3-7. These results demonstrated
that the SIN
isolates described in this study were derived from
the Toto 1101-based
SIN sequence and that compensatory amino acid
coding changes within the
protein-coding regions or base changes
within noncoding regions are not
essential to restore biological
activity to SIN genomes carrying AU
additions or deletions in
their genome termini.
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FIG. 5.
Evolution of the 3' proximal region of SIN isolates.
Infected cell cytoplasmic RNA obtained from the indicated passages of
all virus isolates was reverse transcribed with 18TSac and amplified
by PCR with T11200 and 18TSac. The PCR products were purified and
sequenced using the primer T11200. The 3'CSE (1 to 19) and its
remnants are identified by underlining. The bases newly added during
the repair process are identified by uppercase letters. The hyphens
denote nucleotide identity. Slashes and vertical lines denote
discontinuity in sequence, used for drawing purposes.
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Evolution of the 5' and 3' proximal sequences of various SIN
genomes.
Although the stability and infectivity of these novel SIN
isolates did not require any compensatory base changes in the SIN genomes, it is possible that these genomes may evolve into new and more
efficient genomes. Since the 5' and 3' proximal regions were believed
to carry RNA motifs that regulate RNA synthesis, we analyzed the base
changes in these regions during passage in BHK cells. To determine the
sequences of the 5' proximal region of SIN genomes, the infected cell
cytoplasmic RNA corresponding to p-1 and p-18 of all isolates and a
control SIN RNA corresponding to Toto 1101-RR were reverse transcribed
with the primer T1050, and the resulting first-strand cDNA was tailed
with dCTP and amplified by PCR. The PCR products were purified and
sequenced using primers T200, T500 and T1050 (Table 3). The first
962 bases corresponding to the 5' proximal region of all isolates
corresponding to p-1 and p-18 were identical (data not shown). As
expected, nt 353 was found to be a U in all the isolates, including
that of Toto 1101, whereas the HRsp strain carried a C residue in that
position (58).
Since the 3'NTR domain of these isolates underwent various extents of
the repair process during their generation in the RNA-transfected
cells, we anticipated additional base changes during evolution.
Therefore, we chose to examine virus-specific RNA derived from
passages
1, 2, 6, 10, and 18 of all isolates (Fig.
5). Four of
the isolates,
S3-7, S3-11, S3-10, and S3-20, did not undergo any
base changes within
the terminal 470 bases in any of the passages
tested. Isolate S3-8,
which carried an intact 3'CSE and an insertion
of a short motif, UAUUU,
within the poly(A) tail, underwent deletion
events during passages 3 to
6, resulting in the formation of wild-type
3'NTR. This deletion event
could have occurred by a simple polymerase
skipping event during
synthesis of new RNA. Alternatively, the
initiation of RNA synthesis
could have occurred just upstream
of this UAUUU motif, using the short
oligo(A) motif and the 3'CSE
as a promoter. Isolate S3-9, which carried
only 13 of the 19 bases,
acquired the motif CAUUAC within
its poly(A) tail, and this modified
3' terminus was stable for the
remaining passages as tested. Finally,
isolates S3-4 and S3-23, which
carried extensive AU-rich insertions
just adjacent to the poly(A) tail,
underwent base changes of A
to U. These unusually long 3'NTRs showed
little or no tendency
to reduce their size or composition during
multiple passages.
These results demonstrated the long-term stability
and functional
integrity of different types of 3' AU-rich terminal
elements of
the genomes of the SIN isolates in BHK
cells.
RSEs in the 3'NTR of the new SIN isolates.
Previous work from
other laboratories demonstrated the presence of RSEs within the 3'NTR
(43, 45), but the occurrence of RSEs at other regions of the
alphavirus genome has not been reported. Given the multitude of 3'
terminal motifs that are compatible with efficient SIN genome
replication, the formation and function of the RSEs of 3'NTRs may have
a direct relationship with genome repair. To evaluate the significance
of the RSE in the repair and evolution of alphavirus genomes, the
occurrence and distribution of RSEs within the 5' and 3' proximal
regions of all the isolates were determined. Although numerous RSEs of
4 to 7 bases occurred within the genomes of the SIN isolates, those
motifs which contained eight or more bases were chosen for analysis. As
shown in Table 4, RSE2, RSE3, RSE4, RSE5,
RSE7, and RSE8 were found in duplicates within the 3'NTR of all SIN
isolates studied. RSE1 was found in both the 5' and 3' proximal regions
of all SIN isolates studied. RSE6 was found in duplicate copies only in
the 5' proximal region of all the SIN isolates. As shown in Table 4,
the 3'NTR of the genomes of isolates S3-4 and S3-23 carried many new
additional RSEs. Four of these RSEs (k, l, m, and n), corresponding to
the isolate S3-4, were found in both p-1 and p-10 genomes. The RSEo and
RSEp were found only in the p-10 genome of the isolate S3-4. Likewise,
the 3'NTR of the p1 and p10 genomes of the isolate S3-23 also carried
six RSEs (r, s, t, u, v, and x) whose sequences were different from
that of the isolate S3-4. The p-10 genome of the isolate S3-9 carried
one new RSE (z) within its 3'NTR. The genomes of isolates S3-7, S3-8,
S3-10, S3-11, and S3-20 did not undergo any AU additions during their
biogenesis (46) and therefore did not carry any of the
additional RSEs found in the isolates S3-4 and S3-23. The arrangement
and order of the RSEs found in the genomes of isolates S3-4 and S3-23
are shown in Fig. 6. Detailed analysis
revealed that the entire AU-rich region that replaces the 3'CSE was
composed of RSEs of 4 nt or more (data not shown). Therefore, it is
likely that the 3' repair pathway involving the extensive addition of
AU-rich motifs may involve reiterative copying of short AU-rich motifs
from within the SIN genome.
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FIG. 6.
Physical map of the AU-rich RSEs. Each RSE found within
the AU-rich terminal motif of isolates S3-23 (A) and S3-4 (B) was
arranged and identified by the same letters as indicated in Table 2.
Bars of the same kind indicate the same RSE. For example, bars denoting
the motif x (panel A) occur very close to each other, whereas those
denoting p (panel B) are well separated.
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Kuhn et al. reported that some mutations within the 3'CSE of the SIN
genome were lethal (
32). Results of previous studies
by
members of our group (
24,
46) and the results reported
here
establish that SIN isolates lacking the classical 3'CSE and
isolates
carrying novel AU-rich motifs are highly infectious for
all the 18 passages tested in BHK cells. Increased RNA transfection
efficiencies
and the nature of transfection methodologies adopted
by us might have
helped us to recover these novel virus isolates.
Despite the
differences in their 3' terminal sequences, most of
the isolates showed
very similar growth patterns in BHK cells.
It is puzzling to note that
the isolate S3-20, which lacked 13
bases of the 3'CSE, displayed a
drastic decrease in virus release
during the early phase of infection,
whereas other isolates, such
as S3-9, which lacked 6 bases of the
3'CSE, displayed a normal
rate of virus release. The decreased rate of
virus release could
be due to an early defect in virus assembly or to
decreased levels
of virus-specific RNA during the first 3 to 6 h
of infection.
Although the steady-state levels of virus-specific RNA
were similar
for all the isolates tested, it is possible that the time
course
of RNA synthesis and decay differ for different isolates. The
growth properties and virus-specific RNA levels of isolates S3-4
and
S3-23, which carry extensive AU additions at the 3' end of
their
genomes, were comparable to that of the S3-7 isolate, which
carries the
wild-type 3'NTR. Preliminary results indicated that
even the kinetics
of RNA synthesis was similar among these three
isolates (unpublished
data). Therefore, it is possible that the
classical 3'CSE, the deletion
versions of 3'CSE, as found in the
isolates S3-9, S3-10, and S3-11, and
the AU-rich motifs, as found
in the isolates S3-4 and S3-23, serve as
equally strong promoters
for RNA synthesis. How the SIN polymerase
could initiate RNA synthesis
from a disparate set of 3' termini remains
to be determined. As
described for picornaviruses (
19,
38,
40) and coronaviruses
(
50), an internal or 5' RNA
motif of the alphavirus genome may
be important for genome replication.
We propose that this alternate
RNA motif yet to be identified may
recruit the polymerase to the
SIN genome in the absence of an authentic
3'CSE. Once the polymerase
is recruited to the SIN genome, 3' end
recognition may occur by
polymerase movement on the template RNA. The
3' poly(A) tail,
AU-rich motifs, and proteins associated with these
sequences may
serve as signals for initiation of RNA synthesis once the
polymerase
is selectively loaded on the
template.
No base changes were found in the genomes of five of eight isolates
studied. Importantly, isolates S3-4 and S3-23, which carried
long
AU-rich 3' terminal motifs in their genomes in all of the
18 passages,
were also found to carry no base changes elsewhere
in the genome. Given
the rapid evolution of RNA genomes (
25,
31) and the strict
conservation of the 3'CSE of the alphavirus
genome, it is difficult to
reconcile the fact that there are no
compensatory base changes or
evolutionary pressures for acquiring
the classical 3'CSE. The simplest
explanation is that the 3'CSE
or the AU-rich elements do not bind
directly to the viral polymerase.
A case in point is that the highly
conserved 98-nt 3' terminal
motif of hepatitis C virus failed to bind
to the viral polymerase,
whereas a 3'-coding region with a conserved
stem-loop structure
specifically bound to the viral polymerase
(
11). One or more
of the known RNA binding proteins
(
15,
56) may bind to these
different 3' terminal sequences,
and the viral polymerase may
be recruited to the 3' terminus of the
alphavirus genome through
interaction with these cellular proteins.
Since several AU-rich
3' motifs were able to support RNA synthesis, RNA
binding proteins
are expected to possess a broad specificity for
adenylates and
uridylates, and all of these AU-binding proteins must
carry a
common motif that binds to the viral polymerase. Since
mosquitoes
are vectors for alphaviruses, it is also likely that these
broad-specificity
RNA binding proteins are present in invertebrates. It
is likely
that infectious cycles involving vertebrate and invertebrate
cells
may be essential to restore the classical 3'CSE during virus
passage.
What are the determinants that regulate repair and rearrangement of
sequences within the 3'NTR? The evolution of the 3'NTR
of the SIN
genome of isolate S3-8 indicates that in the presence
of an intact
3'CSE, foreign motifs inserted within the poly(A)
tail are removed,
probably by polymerase jumping events (
6,
28,
34). The 3'
terminal base preceding the poly(A) tail is
a C residue in all
alphaviruses (
45). The 3' terminal C of the
3'CSE, although
not essential for virus replication, appears to
serve as a 3' boundary
for the alphavirus genome. For example,
the absence of the terminal C
residue in the isolate S3-11 might
be responsible for allowing the
3'NTR to retain the stretch of
U residues as part of the genome (Fig.
5C). Similarly, the absence
of the 3'CSE appears to have caused
retention of the long AU-rich
motifs in isolates S3-4 and S3-23 (Fig.
5G and H). SIN genomes
lacking the 3' terminal bases of the 3'CSE
actually stimulated
acquisition of AU-rich motifs (
46).
Isolate S3-9, which lacked
six bases from the 3' terminus of the 3'CSE,
acquired a new motif,
CAUUAC, at its 3' terminus during
passage in BHK cells (Fig.
5E).
Our previous studies (
46)
and the work reported here demonstrate
that SIN genomes carrying
several different 3'NTRs with and without
the classical 3'CSE replicate
as well as wild-type SIN. Deletion
versions of the 3'CSE, as are found
in genomes of the isolates
S3-9 and S3-20, were fully functional even
in the absence of any
RNA repair or 3' terminal AU additions (Fig.
1B).
In vivo processed
and repaired SIN genomes, such as S3-4 and S-23,
which carried
extensive AU-rich motifs at their 3' ends, replicated
efficiently
and maintained these AU-rich motifs through all the 18 passages
tested (Fig.
5G and H). These results argue that repair of the
3' terminus of the 3'NTR of the SIN genome with AU-rich motifs
may be
coincidental to events such as RNA transport and localization
and that
the addition of new AU-rich motifs is not required for
genome
replication. As proposed in a previous section, an alternate
RNA motif
within the SIN genome might serve to recruit the viral
polymerase, and
the modified 3' termini described for the genomes
of these new SIN
isolates may serve as accessory signals that
indirectly regulate genome
replication. The 3'NTR of many eucaryotic
mRNAs carry AU-rich domains,
which are thought to regulate RNA
stability, localization, and
translation (
9,
15). How the
addition or the removal of
AU-rich elements precisely affects
the biology of the SIN replication
remains to be
determined.
The RSEs found in the 5' and 3' proximal regions of the SIN genome may
regulate RNA synthesis. Duplication of these motifs
clearly occurs at a
much higher frequency within short 5' and
3' proximal regions. Since
polymerase jumping and recombination
events are well known among RNA
viruses (
6,
28,
34), including
alphaviruses (
21,
24,
47,
64), these motifs might have
been copied by the viral
polymerase and introduced in the growing
polynucleotide chain. Since a
major portion of newly formed 3'
terminal motifs of isolates such as
S3-4 and S3-23 (Fig.
6) was
composed of 85 to 95% AU-rich RSE, it is
likely that formation
of AU-rich RSEs is due predominantly to viral
polymerase activity.
It appears that the AU-rich domains within the
3'NTR or other
parts of the genome are copied during synthesis of new
RNA to
repair the defective 3' termini of the SIN genomes. Other motifs
containing G or C residues may also be copies for repair purposes.
In
fact, the new motif CAUUAC that was inserted within the
poly(A)
tail during the later passages of the isolate S3-9 (Fig.
5E)
could
have been copied from four different locations (nt 2661, 9218,
9702, and 10202) of the translatable region of the SIN genome.
Although
the size and composition of RNA motifs copied from within
the SIN
genome (or from cellular RNA) to repair the 3' end of
the SIN genome
are not known, it is clear that all of the SIN
isolates that have
undergone repair maintain an AU-rich 3' proximal
region and a poly(A)
tail. It is possible that many different
motifs could have been
introduced at the site of repair, but the
resultant genomes evolve
rapidly within the transfected BHK cell
to acquire the observed 90 to
95% AU-rich 3' terminus. In this
context, it is interesting that the
53-nt 3' terminal region,
excluding the poly(A) tail, of the wild-type
SIN genome is 87%
AU-rich. The 3'CSE, which is highly conserved in all
of the 27
alphaviral genomes, carries an 85 to 90% AU-rich sequence
(
45).
Therefore, it appears that the 3'NTR of the alphavirus
genome
and its repair and the replication machinery have coevolved to
maintain the AU-rich 3' terminus. In contrast, the 3'NTR of rubella
virus, which carries little or no AU-rich 3' terminus, undergoes
a 3'
repair process involving few or no AU-rich terminal additions
(
10). The telomerase activity (
5) of eucaryotic
cells, which
adds repeat sequence elements to the termini of
chromosomes, and
the alphavirus 3' repair machinery, which adds repeat
sequence
elements to the 3' end of the genome, may have evolved to
accomplish
similar
functions.
Several forms of RNA editing (
3) and 3' repair of viral RNA
genomes (
4,
8,
30,
42,
49,
52,
61) have been
documented. It
is possible that these various RNA-modifying machineries
may have
common structural and functional domains (
14). In fact,
an
AU-rich sequence element in the 3'NTR of apolipoprotein B mRNA
was
shown to bind to the classical RNA editing enzyme Apobec-1
(
1). The 3' repair processes observed in the SIN genome
constitute
the only known example that involves de novo polyadenylation
and
addition of different sizes of AU-rich motifs (
46). Both
rubella
virus (
10) and beet necrotic yellow vein virus
(
30) appear
to add a short stretch of U residues adjacent to
the poly(A) tail.
The size and kind of repeat motifs added to the 3'
terminus of
RNA genomes may be regulated by the sequence of the
template RNA
and the composition of the repair machinery, including the
polymerase.
Although many cellular RNA binding proteins (
11,
55,
56),
such as poly(A) binding proteins and polypyrimidine binding
proteins,
may play a role in transport, localization, repair, and
replication
of the SIN genome RNA, the enzymatic activity that
introduces
AU-rich motifs on the 3' terminus of the genome is likely to
involve
the viral RNA-dependent RNA polymerase. As proposed for some
eucaryotic,
procaryotic, and RNA viral systems (
22,
27), the
SIN polymerase
complex, along with the nascent RNA, may undergo
sliding, jumping,
and stuttering events during new RNA synthesis,
resulting in the
introduction of novel motifs during chain elongation
and 3' end
formation. Identification and characterization of the RNA
motifs
and protein factors that regulate alphavirus genome repair may
give new insights into the mechanism of template selection in
virus-infected
cells.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant GM 57439.
We acknowledge the use of the DNA Sequencing Core Facility of the
Vanderbilt-Ingram Cancer Center. We thank Joel Trupin, Kolari Bhat, and
Andy Briscoe and members of our laboratory for critical reading of the
manuscript and fruitful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Rm. 4126, Basic Sciences Building, 1005 D. B. Todd Blvd., Meharry Medical College, School of Medicine,
Nashville, TN 37208. Phone: (615) 327-6687. Fax: (615) 327-6602. E-mail: rramasamy{at}mmc.edu.
| |
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Journal of Virology, October 2000, p. 9776-9785, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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