Previous Article | Next Article 
J Virol, January 1998, p. 10-19, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Functional Antigenomic Promoter for the
Paramyxovirus Simian Virus 5 Requires Proper Spacing between an
Essential Internal Segment and the 3' Terminus
Susan K.
Murphy,1
Yasuhiko
Ito,2 and
Griffith D.
Parks1,*
Department of Microbiology and Immunology,
Wake Forest University Medical Center, Winston-Salem, North
Carolina 27157-1064,1 and
Department of
Microbiology, Mie University School of Medicine, Edobashi, Tsu-Shi,
Mie-Ken, Japan2
Received 10 July 1997/Accepted 26 September 1997
 |
ABSTRACT |
A previous analysis of naturally occurring defective interfering
(DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5)
indicated that 113 bases at the 3' terminus of the antigenome were
sufficient to direct RNA encapsidation and replication. A nucleotide
sequence alignment of the antigenomic 3'-terminal 113 bases of members
of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at
the 3' terminus (conserved region I [CRI]) and a more distal region
consisting of antigenome bases 73 to 90 (CRII) that was contained
within the 3' coding region of the L protein gene. To determine whether
these regions of the antigenome were essential for SV5 RNA replication,
a reverse genetics system was used to analyze the replication of
copyback DI RNA analogs that contained a foreign gene (GL, encoding
green fluorescence protein) flanked by 113 5'-terminal bases and
various amounts of SV5 3'-terminal antigenomic sequences. Results from
a deletion analysis showed that efficient encapsidation and replication
of SV5-GL DI RNA analogs occurred when the 90 3'-terminal bases of the
SV5 antigenomic RNA were retained, but replication was reduced ~5- to
14-fold in the case of truncated antigenomes that lacked the 3'-end
CRII sequences. A chimeric copyback DI RNA containing the 3'-terminal 98 bases including the CRI and CRII sequences from the human
parainfluenza virus type 2 (HPIV2) antigenome in place of the
corresponding SV5 sequences was efficiently replicated by SV5
cDNA-derived components. However, replication was reduced ~20-fold
for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII
sequences between antigenome bases 72 and 90. Progressive deletions of
6 to 18 bases in the region located between the SV5 antigenomic CRI and
CRII segments (3'-end nucleotides 21 to 38) resulted in a ~25-fold
decrease in SV5-GL RNA synthesis. Surprisingly, replication was
restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3' terminus. A model is presented for the
structure of the 3' end of the SV5 antigenome which proposes that
positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.
| |
INTRODUCTION |
The paramyxoviruses are a diverse
family of nonsegmented negative-sense RNA viruses that includes Sendai
virus (SeV), measles virus (MeV), and respiratory syncytial virus
(RSV). Simian virus 5 (SV5) is a prototype of the
Rubulavirus genus of the Paramyxoviridae family, which includes mumps virus (MuV), SV41, and human
parainfluenza virus type 2 (HPIV2). The ~15-kb paramyxovirus
genomic and antigenomic RNAs are tightly bound by the viral
nucleocapsid protein (NP) to form nucleocapsid (NC) structures, and it
is these ribonucleoprotein complexes that serve as the only templates
for viral RNA synthesis. The viral phosphoprotein (P) and the large
protein (L) together form the viral RNA-dependent RNA polymerase which
is responsible for both transcription to produce mRNAs and replication
to produce negative-sense genomes and positive-sense antigenomes
(18, 20, 24). For the nonsegmented negative-sense RNA
viruses, RNA synthesis initiates from promoters located at the 3' ends
of both the genomic and antigenomic RNAs, but understanding of critical
features of the viral template that control the level and particular
type of RNA synthesized by the viral polymerase is incomplete.
Two factors that can influence the level of RNA synthesized from the
paramyxovirus antigenomic promoter have been described: the total
number of nucleotides in the viral RNA and the primary nucleotide
sequence at the termini of the viral RNA. The length of a paramyxovirus
RNA can be a major factor that determines the level of RNA replication,
with genome replication being most efficient when the total number of
nucleotides is an even multiple of six (6). The rule of six
is thought to reflect critical interactions between the viral
polymerase and 3'-terminal promoter sequences, with initiation of RNA
replication being most efficient when the last six bases of the 3'-end
promoter are bound completely by a single NP (6, 35). The
degree to which replication of a particular paramyxovirus genome
adheres to the rule of six differs among viruses. For SeV, the rule of
six is an apparent strict requisite (6), while RSV shows no
particular replicative advantage for genomes having any of the integer
lengths tested (41). SV5 is unique in this regard, since the
stringency of the rule of six for SV5 defective interfering (DI) RNA
replication is intermediate between that found previously for SeV and
RSV (26).
A second major factor in paramyxovirus RNA replication is the primary
sequence at the 3' and 5' termini of the viral RNA, as shown previously
by mutagenesis studies (44) and by the structure of
paramyxovirus DI RNAs. These subgenomic RNAs contain various segments
from the standard nondefective viral genome that are characteristic of
an individual DI RNA. However, all naturally occurring DI genomes that
are competent for replication retain either the genomic 3' and 5' ends
in the case of internal deletion of DI RNAs or the genomic 5' end and
its complement for copyback DI RNAs (reviewed in reference
36). The nucleotide sequence in the leader (le) RNA
at the 3' terminus of the genome and in the antitrailer (tr') RNA at
the 3' terminus of the antigenome have been proposed to be primary
determinants of the efficiency of paramyxovirus replication to produce
antigenomes and genomes, respectively (7, 44). The levels of
RNA synthesized from these two viral promoters are reported to differ
significantly (7, 22, 44, 46), but the basis for these
differences in promoter activity and important features of the
3'-terminal regions is not completely understood.
The work reported here provides evidence for a third factor that can
dramatically influence the level of RNA synthesized from the
paramyxovirus antigenomic promoter. We have used a reverse genetics
system to analyze features of the 3'-terminal region of the SV5
antigenome that are important for directing RNA replication. The
results indicate that a functional SV5 antigenomic promoter requires
two discontinuous regions (CRI and CRII) that are located in the
3'-terminal 90 bases of the antigenomic RNA. Most importantly, the
relative spacing of these two cis-acting regions is a
critical factor in determining the level of SV5 RNA replication.
Therefore, the overall number of nucleotides in a viral RNA, the
primary nucleotide sequence at the termini, and proper spacing of
promoter elements are three features of the viral RNA template that can significantly influence paramyxovirus RNA synthesis. We present a model
for the structure of the 3' end of the SV5 antigenome which proposes
that positioning of CRI and CRII along the same face of the helical
nucleocapsid is an essential feature of a functional antigenomic
promoter.
| |
MATERIALS AND METHODS |
Cells and viruses.
Monolayer cultures of A549 cells were
grown and infected with virus as previously described (26).
Vaccinia virus vTF7.3 (14) was grown and titered in CV1
cells.
Nomenclature.
DNA plasmids encoded antigenomic RNAs flanked
on the 3' and 5' ends by the hepatitis delta virus (HDV) ribozyme and
the T7 RNA polymerase (T7 pol) promoter, respectively (26, 32,
34). Plasmids encoding SV5 DI RNA analogs are named to denote
three portions of the antigenome: the 5' end (proximal to the T7
promoter), the internal segment flanked by complementary termini, and
the 3' end (proximal to HDV ribozyme). Thus, the 113-GL-59 antigenomic RNA contains 113 SV5-specific 5'-terminal nucleotides linked to the
Green Lantern (GL; Gibco BRL) open reading frame followed by 59 SV5-specific 3'-terminal nucleotides. Likewise, the DI-SSP antigenomic
RNA contains SV5 sequences at the 5' end (S) and internal position (S),
linked to HPIV2-specific 3' antigenomic RNA sequences (P).
Construction of plasmids encoding the SV5 antigenomes.
All
viral genomes were designed to maintain an overall 6N length. PCR was
carried out as described previously (30), using Pwo polymerase (Boehringer Mannheim, Indianapolis,
Ind.) along with oligonucleotides listed in Table
1. The sequences of all PCR-derived DNA
segments were confirmed by nucleotide sequence analysis (1).
The cDNAs encoding copyback genomes DI852 and DI499+5 (DI504) have been
described previously (26). To construct deletion mutant
DI462, the 5' antigenomic region of terminal complementarity in pDI852
was amplified in a PCR along with the 462 primer (Table 1) and a primer
specific for the T7 promoter sequence in pDI852 (Fig.
1A). The resulting product was digested
with EcoRI and cloned into the PvuII and
EcoRI sites of pGem3term (a pGem3 plasmid previously modified to contain the T7 terminator sequence downstream from the SP6
promoter [26]. The EcoRI-SphI
fragment encoding the 3' antigenomic end of pDI852 (see Fig. 1 for
schematic) was then inserted into the corresponding sites of the
subclone to yield pDI462.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1.
Sequences located internal to the 113-nucleotide
terminal complementary regions of DI852 are not required for
replication. (A) Structures of SV5 DI genomes DI852, DI504, and
DI462. The cDNAs encoding SV5 DI genomes are shown schematically as
rectangles, with cross-hatched boxes and lines representing the
complementary termini and internal deletions, respectively. The
promoter for T7 pol (T7p), the self-cleaving HDV ribozyme ( ), and T7
terminator sequence (ø) are indicated for DI852 only but are included
in all cDNAs encoding SV5 DI RNA analogs. Numbers that denote
nucleotides composing the 5' region of terminal complementarity (113 bases), the 3' end of the L gene (708 bases), the 31-base tr', and
internal deletions are indicated. The horizontal arrow indicates the
direction of transcription from the T7 pol promoter to produce a
positive-sense DI RNA antigenome containing three nonviral G residues
(ggg) at the 5' end. Replication levels are expressed as percentages
(±SD) of that determined for the WT DI852 assayed in parallel. (B)
Replication of DI852, DI504, and DI462 genomes. A549 cells were
infected with vTF7.3 and then cotransfected with plasmids encoding the
L (2.0 µg), P (0.2 µg), and NP (2.0 µg) proteins along with an
individual DI antigenome (1.0 µg). Total intracellular RNA was
harvested and analyzed by Northern blotting with a
32P-labeled positive-sense riboprobe corresponding to
sequences located internal to the 113 bases of terminal
complementarity.
|
|
Copyback DI RNA analogs containing a foreign gene were constructed by
linking DNA fragments from independent PCRs such that
a minus-sense
copy of the GL open reading frame was inserted between
various amounts
of SV5-specific 5' and 3' termini. For the purposes
of nomenclature,
the primary T7 transcripts generated from this
series of constructs are
designated antigenomes and have the SV5
termini in the antigenomic
sense flanking the GL segment. To generate
cDNAs encoding DI genomes
with various amounts of the SV5 termini,
we used a three-step cloning
procedure that involved the use of
primers (P113, P90, P72, P59, and
P31 [Table
1]) capable of annealing
to opposite strands of both
termini of pDI852. However, specific
amplification of a single terminus
occurs when these primers are
used in conjunction with either the SP6
or T7 primer. First, the
P113 and P59 primers were used with the T7
primer on the pDI852
template to generate DNA products encoding the
antigenomic 113
or 59 SV5 5'-terminal nucleotides. The PCR products
resulting
from amplification with the T7-P113 and T7-P59 primer
combinations
were digested with
BamHI and cloned into the
PvuII and
BamHI sites
of pGem3term to yield
pGem3-113 and pGem3-59. In the second step,
the GL open reading frame
contained in the pGreen Lantern-1 DNA
template (Gibco BRL) was
amplified in a PCR with oligonucleotides
GL-ATG and GL-TGA. The
resulting GL-specific product was cloned
into the
EcoRI and
StuI sites of pGem3-113 and pGem3-59 to give
pGem3-113-GL
and pGem3-59-GL, respectively. In the third step,
which generated the
3' antigenomic termini of the new constructs,
plasmid pDI852 was used
as the template in a PCR with primer P113,
P90, P72, P59, or P31 and a
primer specific for the SP6 promoter,
which is located between the
SphI site and T7 terminator sequence
of pDI852 (Fig.
1A).
The
StuI-
SphI fragments resulting from PCRs
using
the SP6-P113, -P90, -P72, -P59, and -P31 primer combinations
were then
ligated into the corresponding sites of pGem3-113-GL
to generate the
full-length constructs pDI113-GL-113, -90, -72,
-59, and -31. Similarly, the
StuI-
SphI fragment from the
P113-SP6
PCR was ligated into the corresponding sites of pGem3-59-GL to
produce pDI59-GL-113.
Nucleotide replacements, insertions, or deletions were engineered into
SV5 3' antigenomic segments by using oligonucleotide-directed
mutagenesis as previously described (
30). Plasmid pDI852 had
been previously modified by using the Tr'Bam and SP6 primers to
contain
a
BamHI site (5'-GGATCC) in place of bases 39 to
44 from
the 3' end of the antigenomic RNA. pDI852 containing the
BamHI
site was used as the template in a PCR with the SP6
and P90 primers.
The products were digested with
StuI and
SphI and cloned into
the corresponding sites of pDI113-GL-90
to generate pDI113-GL-90B
(see Fig.
7A). pDI113-GL-90B was used in the
generation of the
length-altered and nucleotide replacement mutants.
The pDI113-GL-90B
6, -90B
12, -90B
18, -90BRe12, -90BRe18,
-90B+6, or -90B+12 antigenome contained a deletion of 6, 12, or
18 nucleotides (antigenomic bases 33 to 38, 27 to 38, or 21 to
38), a
replacement of 12 or 18 nucleotides (antigenomic bases
27 to 38 or 21 to 38), or an insertion of 6 or 12 nucleotides
between antigenomic
bases 38 and 39, respectively. These altered
SV5 antigenomic 3'
segments were prepared by using pDI113-GL-90B
as the template in a PCR
along with the SP6 primer and primer
Tr'del6, Tr'del12, Tr'del18,
Tr're12, Tr're18, Tr'ins6, or Tr'ins12.
The products were digested with
BamHI and
SphI and cloned into
the corresponding
sites of pDI113-GL-90B to generate the full-length
constructs. The
pDI113-GL-90Re25 construct contained a replacement
of 25 nucleotides at
antigenomic bases 14 to 38. The Tr're25 primer
was used with the SP6
primer in a PCR on template pDI113-GL-90B,
and this PCR product was
used as a megaprimer (
30) in a second
PCR for five cycles
along with primer GL-1 on template pDI113-GL-90BRe18
(described above).
These DNAs were then amplified in a third PCR
with the SP6 and GL-1
primers. The resulting products were digested
with
StuI and
SphI and cloned into the corresponding sites of
pDI113-GL-90.
To construct chimeric SV5-HPIV2 copyback DI antigenomes, a
BamHI fragment encoding the 3' end of the HPIV2 antigenome
(L protein
base 5929 to the 3' terminus [
19]) was
excised from pBSIISK-HPIV2Lpro
(
19) and subcloned into the
BamHI site of pGem3 to yield pG3-P2L.
To construct pDI-PSS,
a new T7 pol promoter was fused to the HPIV2
5' terminus in a PCR using
pG3-P2L as the template along with
the HPIV2T7 and T7 primers. The PCR
product was digested with
Asp718 and
EcoRV and
ligated into the corresponding sites of pDI852
before linking with the
SspI to
SphI fragment encoding the SV5
3'-terminal antigenomic sequences and HDV ribozyme described previously
(
26). Plasmid pDI-PSS contained (5' to 3') the T7 pol
promoter,
HPIV2 5' genomic sequence from positions 1 to 98, a
three-base
linker, SV5 L gene sequences from bases 6121 to 6859 (
31), including
the tr', and the self-cleaving HDV ribozyme.
To construct pDI-SSP,
pDI852 was used as the template in a PCR along
with HPIV2ribo
and SP6 primers. The PCR product then served as a
megaprimer (
30)
along with the T7 primer in a PCR using
pG3-P2L as the template.
The product DNA was digested with
EcoRV and
SphI and ligated into
the corresponding
sites of pDI852, before linking with the
SspI-to-
EcoRV
fragment that encodes the SV5
5'-terminal sequences from the wild-type
(WT) DI852 genome
(
26). Plasmid pDI-SSP contained (5' to 3')
the T7 polymerase
promoter, SV5 DI852-specific sequences up to
base 6752 of the L protein
gene (
26), the 98 3'-terminal bases
from the HPIV2
antigenomic RNA including the tr', and the self-cleaving
HDV ribozyme.
To generate a chimeric DI RNA in which HPIV2-specific
CRII had been
deleted, pDI-SSP was used as template in a PCR along
with the HPIV2del
and SP6 primers. The DNA fragment was digested
with
EcoRV
and
SphI and reconstructed into pDI852 as described
above to
yield pDI-SSPt. This plasmid differs from the parental
pDI-SSP by
containing a three-base linker separating the 3'-terminal
71 bases of
the HPIV2 antigenomic RNA from SV5 sequences.
Analysis of in vivo DI RNA replication from cDNA-derived
components.
A549 cells in 3.5-cm-diameter dishes were infected
(multiplicity of infection of ~5) for 1 h with vTF7.3
(14) and then cotransfected with DNA encoding the SV5
antigenomes (1.0 µg) along with pGem3-L (2.0 µg), pGem2-P (0.2 µg), and pUC19-NP (2.0 µg) as described previously (26),
using a cationic liposome reagent (40). pGem control plasmid
was used to normalize for the amount of transfected DNA. Total
intracellular RNA (~20 µg) was harvested at ~40 to 48 h
posttransfection and analyzed by Northern blot analysis as previously
described (26).
A positive-sense
32P-labeled riboprobe corresponding to
bases 6121 to 6752 of the SV5 L protein gene was generated from plasmid
pGEM5-
SspI for use in analyzing replication of DI852-derived
antigenomes
(
26). For analyzing replication of DI RNAs
carrying the GL sequence,
the DNA fragment derived from a PCR with
primers GL-ATG and GL-TGA
and the pGreen Lantern-1 template described
above was digested
with
EcoRI and
StuI and cloned
into the corresponding sites of
pGem2 (Promega, Madison, Wis.) to
generate pGem2-GL. A 219-base
riboprobe of antisense polarity (which is
of the same polarity
as the GL sequence contained in the T7 primary
transcripts of
the SV5-GL RNAs) was generated from
BamHI-linearized pGem2-GL
by SP6 RNA polymerase in the
presence of [
32P]CTP. For detecting primary T7
pol-derived RNA transcripts, a
positive-sense 782-base
32P-labeled riboprobe was generated by using T7 pol from
the same
template that was linearized with
EcoRI. Membranes
were hybridized
with purified riboprobes (~10
6 cpm)
in ExpressHyb (Clontech, Palo Alto, Calif.) for 1 h at 68°C.
After washing (15 min each in 2 × SSC (1 × SSC is 0.15 M
NaCl
plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate
[SDS],
0.2 × SSC-0.1% SDS, and 0.1 × SSC-0.1% SDS),
the membrane was
exposed at
70°C to radiographic film. Quantitation
of RNA replication
products was performed with the AMBIS 4000 image acquisition system
and software (AMBIS, San Diego, Calif.), with
values reported
in the figures representing the means from at
least three independent
experiments ± standard deviations
(SD).
For analysis of RNAs from CsCl gradients, duplicate 3.5-cm-diameter
dishes of vTF7.3-infected A549 cells were transfected
as described
above. At 40 h posttransfection, cell lysates were
prepared in 0.5 ml of NTE buffer (0.15 M NaCl, 50 mM Tris [pH
7.4], 10 mM EDTA)
containing 0.5% Nonidet P-40 and clarified by
centrifugation (5 min,
14,000 × g), and 400 µl of the resulting
supernatant was
layered onto preformed 20 to 40% (wt/wt, in NTE)
CsCl gradients. After
centrifugation (4 h, 45,000 rpm, 12°C, SW50.1
rotor), samples from
the 30% CsCl fraction were collected (
10),
diluted with
NTE, pelleted (6 h, 35,000 rpm, SW41 rotor), and
analyzed by Northern
blotting as described above.
| |
RESULTS |
Sequences located internal to the 113-nucleotide termini of
naturally occurring SV5 DI RNAs are not required for replication in
vivo.
DI852 is an 852-base copyback DI RNA that was generated
during serial undiluted passage of SV5 in tissue culture
(26). As shown in Fig. 1A, the DI852 antigenome
contains a 113-base 5' region of terminal complementarity linked to
708 bases from the L protein gene and the 31-base 3' tr'. DI499 is
identical to DI852 with the exception of a 353-base deletion of
sequences internal to the 113-base region of terminal complementarity.
A five-base insertion into DI499 created a 6N-length antigenome
(DI499 + 5 or DI504 [Fig. 1A]) and enhanced replication
~10-fold (26). This previous result suggested that the
353-base internal deletion in DI499 had not removed a
cis-acting sequence that was essential for RNA
replication. To determine if the remaining internal sequences in common
between DI852 and DI504 are required for SV5 RNA replication, this
region was deleted to create pDI462, which encoded an antigenomic RNA
with a 390-base deletion (Fig. 1A). DNA segments encoding the
full-length DI852 or internally deleted DI499 and DI462 RNAs were
inserted between the promoter for T7 pol and the self-cleaving HDV
ribozyme (Fig. 1A, T7p and HDV, respectively) such that transcription from the T7 pol promoter would generate a positive-sense
antigenomic RNA containing three additional 5'
guanosine (G) residues and an exact 3' end due to
ribozyme self-cleavage (32, 34, 37).
To determine the relative levels of replication for these SV5 DI RNAs,
monolayers of A549 cells were first infected with vTF7.3
(
14), a recombinant vaccinia virus that expresses the
bacteriophage
T7 pol. These infected cells were then cotransfected with
plasmids
encoding the L, P, and NP proteins along with one of the SV5
antigenomic
RNAs, each of which was under control of the T7 pol
promoter.
Northern blot analysis of total intracellular RNA with a
positive-sense
riboprobe was used to monitor conversion of the
positive-sense
antigenome synthesized by T7 pol to the negative-sense
complementary
strand by the SV5 polymerase. As shown in Fig.
1B, RNA
from cells
cotransfected with the L, P, and NP plasmids and the
copyback
DI852 antigenome contained a major ~0.9-kb RNA species that
was
complementary to the T7 pol-derived RNA transcript, and this RNA
was not detected when plasmids encoding L, P, or NP protein were
omitted (not shown, but see below). In addition to the ~0.9-kb
DI852
replication product, low levels of a ~1.8-kb RNA species
of unknown
origin were detected. The levels of replication for
both of the
internal deletion mutants were similar to that seen
with the WT DI852
(Fig.
1A, 62 and 72% for DI504 and DI462, respectively).
These data
support the contention that sequences internal to the
3'- and
5'-terminal 113 bases of DI852 are not required for SV5
antigenome
replication.
Identification of two regions of sequence identity in the
3'-terminal sequences of the Rubulavirus antigenomic
RNA.
The foregoing results indicated that 113 bases at the 3'
terminus of the SV5 antigenome contain all of the signals necessary for
RNA encapsidation and replication. To determine if sequences contained
in these 3'-terminal 113 bases were conserved among other members of
the Rubulavirus genus of the Paramyxoviridae family, the 3' ends of the SV5, HPIV2, SV41, and MuV antigenomes were
compared. As shown in Fig. 2, the
nucleotide sequence alignment identified two regions of sequence
identity: bases 1 to 19 at the 3' terminus of the antigenome
(CRI) and a more distal region consisting of antigenome bases
73 to 90 (CRII) that was contained within the 3' coding region of the L
protein gene. Sequence identity in CRII did not solely reflect
conservation due to coding of L protein amino acids, since the
corresponding 90- to 72-base region from the MuV antigenome is located
in the 3' noncoding segment of the L gene (28). Likewise,
when the 3' ends of the SV5 antigenomic (L-tr' region) and genomic
(le-NP region) RNAs were aligned (Fig. 2, SV5 le sequence), significant
sequence identity was found in CRI (16 of 19 bases) and CRII (11 of 18 bases). Thus, CRI and CRII show sequence identity among rubulaviruses
and are located at the 3' termini of both the SV5 genomic and
antigenomic RNAs.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 2.
Nucleotide sequence alignment of the SV5, HPIV2, SV41,
and MuV antigenomic 3' termini. The 3'-terminal 113 bases from the
antigenomes of four members of the Rubulavirus genus of
paramyxoviruses (SV5, HPIV2, SV41, and MuV) are listed. Amino acids and
the translational stop codon (boxed) that are encoded in the 3' end of
the SV5 L gene are designated by one-letter abbreviations above the SV5
sequence, and the site for poly(A) addition is underlined. Solid
circles indicate positions of sequence identity between all four
viruses. SV5 le is the 3'-proximal 113 nucleotides of the SV5 genomic
RNA, with the arrowhead at base 56 denoting the start site for
transcription of the NP gene. Asterisks denote positions of sequence
identity between the SV5 genomic le-NP and antigenomic L-tr' regions.
CRI (3' proximal) and CRII (within the SV5 L gene) are indicated by the
shaded boxes encompassing positions 1 to 19 and 73 to 90, respectively.
Vertical bars in the context of the HPIV2 sequence delineate the region
(bases 98 to 72) deleted to generate pDI-SSPt as described in the text.
Sequences (references): SV5 (31); HPIV2 (19);
SV41 (27); MuV (28).
|
|
Efficient replication of copyback DI analogs containing 90 bases
from the 3' terminus of the SV5 antigenome.
To determine if the
113-nucleotide complementary regions of DI852 containing CRI and CRII
were capable of directing the encapsidation and replication of a
foreign reporter sequence, we constructed a plasmid (pDI113-GL-113)
such that a negative-sense copy of the GL open reading frame was
flanked on the 5' and 3' ends by the 113-nucleotide terminal
complementary regions derived from DI852 (Fig.
3A). Plasmid DNA encoding the 113-GL-113
DI RNA (designated an antigenome) was cotransfected along with
the L, P, and NP plasmids into vTF7.3-infected A549 cells. Total
intracellular RNA was analyzed by Northern blotting with a
negative-sense riboprobe specific for the GL sequences contained within
the replication product of the SV5-GL DI RNA analog. As shown in Fig.
3B, RNA from cells transfected with this combination of plasmids
contained a ~990-base RNA that hybridized to the negative-sense GL
riboprobe (arrow, lane 1), and this RNA species was not detected in
control samples from transfected cells in which the L, P, and NP
plasmids had been omitted (Fig. 3B, lanes 2 to 4, respectively). As
noted previously (26), a faster-migrating GL-specific
species was detected in RNA from cells in which the NP plasmid had been
omitted from the transfection (lane 4), but the origin of this RNA is
not known.
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 3.
Efficient replication of copyback DI analogs having 90 bases from the 3' terminus of the SV5 antigenome. (A) Structures
of the SV5-GL DI RNA analogs. Viral antigenomes are shown schematically
as rectangles, with cross-hatched boxes and solid lines representing
SV5-specific bases flanking the GL sequences and deletions,
respectively. The predicted sizes of the DI RNA analogs and replication
level as a percentage (±SD) of that determined for the WT genome
analog (113-GL-113) assayed in parallel are shown. (B) Replication of a
foreign gene by SV5 cDNA-derived components. vTF7.3-infected A549 cells
were cotransfected with plasmids encoding the SV5 L, P, and NP proteins
along with plasmids encoding the SV5 DI113-GL-113 genome analog. Total
intracellular RNA was harvested and analyzed by Northern blotting using
a 32P-labeled negative-sense GL-specific riboprobe of the
same polarity as the T7 pol-derived transcript (lane 1). Lanes 2 to 4 represent the replication products generated in cells in which the
indicated support plasmids were replaced with pGem control DNA. The
arrow indicates the position of full-length replication products. (C)
Efficient replication of SV5-GL DI RNA analogs requires viral sequences
between antigenomic bases 90 and 72. vTF7.3-infected A549 cells were
transfected with plasmids encoding L, P, NP, and the WT SV5-GL DI RNA
analog (lane 1) or RNAs that were altered such that they retained 90, 72, 59, or 31 SV5-specific bases at the antigenomic 3' terminus (lanes
2 to 5, respectively). Replication products were analyzed by Northern
blotting as described above.
|
|
To determine the minimum continuous 3'-terminal antigenomic sequences
that were sufficient for replication of the SV5-GL DI
RNA analog,
progressive 5'-to-3' deletions were engineered into
pDI113-GL-113 to
create antigenomes that contained at their 3'
ends the terminal 90, 72, 59, or 31 bases from the SV5 antigenome
(Fig.
3A). When analyzed in the
DI replication assay outlined
above, an SV5-GL antigenome containing 90 3'-terminal bases replicated
to levels that were equivalent to or
greater than that of the
DI113-GL-113 RNA (Fig.
3C, lane 2;
quantitation in Fig.
3A). By
contrast, the replication of SV5-GL
antigenomes with 72 or 59
bases of viral 3'-terminal sequences was
reduced to ~18% of WT
levels (lanes 3 and 4). Further truncation of
the 3' SV5 sequences
to the 31-base tr' region produced an SV5-GL
antigenome that was
replicated to only ~7% of WT levels. Together,
these data suggest
that segments of the SV5 antigenome located between
bases 90 and
72 and perhaps 59 and 31 are important for RNA
replication. In
the case of the SV5-GL antigenomes containing 72 and 59 SV5 3'-terminal
bases, distinct RNAs of unknown origin that were
smaller than
the full-length DI RNA analog were synthesized.
During replication, paramyxovirus RNA synthesis is tightly coupled to
encapsidation of the nascent RNA chain by the viral
NP protein
(reviewed in reference
20). To determine if RNA
synthesized
from the SV5-GL DI RNA analogs was encapsidated into
NC-like structures,
vTF7.3-infected A549 cells were transfected with
DNA encoding
either the DI113-GL-113, -90, or -59 antigenome, along
with the
L, P, and NP plasmids. Cell lysates were then fractionated by
centrifugation on 20 to 40% CsCl gradients. Samples from the 30%
CsCl
fraction of the gradient were collected and analyzed by Northern
blotting with the same GL-specific riboprobe as described above.
As
shown in Fig.
4, positive-sense
GL-specific RNA was recovered
from the 30% CsCl fraction of the
gradient in the case of the
DI113-GL-90 and DI113-GL-113 antigenomes
(lanes 1 and 3). GL-specific
genome-length RNAs were not detected in
the case of DI113-GL-59
(lane 4) or control samples in which the
plasmid encoding the
SV5 L protein was omitted from the transfected DNA
(lane 2). Less-than-genome-length
positive-sense GL-specific RNAs were
recovered from the DI113-GL-59
CsCl gradient (Fig.
4, lane 4),
suggesting that the small RNA
species detected previously in samples of
total intracellular
RNA (Fig.
3C) were encapsidated by NP. Together
with the results
shown in Fig.
3, these data indicate that 90 nucleotides located
at the 3' terminus of the SV5 antigenomic RNA are
capable of directing
the encapsidation and synthesis of replication
products that have
characteristics of bona fide NCs. However, the
deletion of a segment
containing CRII sequences significantly reduces
SV5 RNA replication.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4.
Replication products from the SV5-GL DI RNA analogs have
characteristics of viral nucleocapsids. A549 cells infected with
vTF7.3 were cotransfected with plasmids encoding the SV5 L, P, and NP
plasmids along with the SV5-GL DI RNA analogs containing the indicated
lengths of SV5-specific 3'-terminal bases. Cell lysates were prepared
and analyzed by CsCl density gradient centrifugation. Samples from the
30% CsCl fraction were collected and analyzed by Northern blotting as
described in the legend to Fig. 3. The arrow indicates the position of
full-length replication products. Lane 2 represents a sample analyzed
in parallel from cells in which the L protein plasmid had been replaced
by pGem control plasmid during the transfection.
|
|
Encapsidation of replication-defective SV5-GL antigenomes that
contain truncated terminal regions.
In the foregoing assay for in
vivo DI replication, RNA antigenomes synthesized by T7 pol
transcription of plasmid DNA must be encapsidated by the viral NP
before functioning as a template for replication by the viral
polymerase (32, 34). Therefore, a possible explanation for
the low replication levels associated with the 3'-terminally truncated
SV5-GL antigenomes is that they are defective in NP-mediated
encapsidation of the primary T7 pol-derived RNA. To examine this
possibility, duplicate dishes of vTF7.3-infected cells were transfected
with the P and NP plasmids along with DNA encoding the 113-GL-113,
113-GL-59, or 59-GL-113 antigenome. One set of dishes received the L
protein plasmid, while the other received pGem control DNA. In the
absence of L protein, it was anticipated that encapsidated antigenomic
RNAs would be derived exclusively from T7 pol-derived transcripts,
while in the presence of L protein, these encapsidated RNAs would also
include replication products generated by the SV5 polymerase. Cell
lysates were prepared and analyzed by CsCl gradient centrifugation.
Samples from the 30% CsCl fraction of the gradient were analyzed by
Northern blotting with a positive-sense riboprobe complementary to the
negative-sense GL sequences in the T7 pol-derived RNA.
In the absence of transfected L protein plasmid, the amount of T7
pol-derived RNA recovered from CsCl gradients did not differ
significantly between the various SV5-GL antigenomes (Fig.
5,
genome RNA species, lanes 2, 4, and
6). A slightly larger GL-specific
RNA was also detected in these
samples (Fig.
5, arrowhead), and
the size of this RNA is consistent
with it being a T7 pol-derived
antigenome containing uncleaved
3'-terminal HDV ribozyme sequences
as described previously for RSV
minigenome analogs (
16). In
the presence of transfected L
protein plasmid, the amount of genome-size
113-GL-113 RNA was enhanced,
while the level of the corresponding
RNA from the 113-GL-59 and
59-GL-113 antigenomes remained relatively
constant. The enhanced signal
obtained with the replication-competent
113-GL-113 antigenome probably
reflects the sum of primary T7
pol-derived RNA synthesis and synthesis
of RNA that results from
the multiple rounds of genome replication by
the SV5 polymerase.
Most important, the 59-GL-113 primary RNA
transcript contains
at the 5' end the same truncation of SV5-specific
terminal bases
as is found at the 3' end of the replication-defective
113-GL-59
antigenome (Fig.
3), and the 59-GL-113 antigenome was
encapsidated
at levels equivalent to those for the constructs
containing the
full 113 nucleotides at the 5' end of the primary
transcript.
These data indicate that the low replication of SV5-GL
antigenomes
lacking CRII sequences cannot be accounted for by
significant
defects in encapsidation of the T7 pol-derived RNA.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
Encapsidation of replication-defective SV5-GL
antigenomes that contain truncated terminal regions.
vTF7.3-infected A549 cells were cotransfected with plasmids encoding
the SV5 P and NP proteins and the indicated SV5-GL RNA antigenome
(113-GL-113, 113-GL-59, or 59-GL-113) in the presence (+) or absence
() of the L plasmid. Cell lysates were prepared and analyzed by CsCl
density gradient centrifugation. Samples recovered from the 30% CsCl
fraction of the gradient were analyzed by Northern blotting using a
GL-specific 32P-labeled riboprobe complementary to the T7
pol-derived transcript. Genome-length RNA (genome) and a species that
corresponds to transcripts with an uncleaved HDV ribozyme (arrowhead)
are indicated.
|
|
Replication of chimeric DI RNAs containing exchanges between HPIV2
and SV5 antigenomic 3'-terminal sequences.
A comparison of SV5 and
HPIV2 3'-terminal antigenomic sequences showed a significant degree of
sequence identity in CRI (89% over bases 1 to 19) and CRII (83% over
bases 73 to 90), whereas the sequence of the intervening region is less
well conserved (40% over bases 20 to 72 [Fig. 2]). A plasmid
encoding an 840-base chimeric DI RNA was constructed to determine if
the HPIV2 antigenomic 3' end, containing the HPIV2 CRI and CRII
sequences, could direct DI RNA replication by the SV5 polymerase. In
DI-SSP, 113 3'-terminal bases of the SV5 antigenome were replaced by 98 bases from the corresponding region of the HPIV2 antigenome (Fig.
6A). A549 cells infected with vTF7.3 were
cotransfected with the L, P, and NP plasmids along with DNA encoding
DI852 (SSS) or DI-SSP. As shown in Fig. 6B, RNA from cells transfected
with plasmid encoding DI-SSP contained a major RNA species with an
electrophoretic mobility closely matching that of DI852 (lanes 1 and
2). The level of replication for the SV5-HPIV2 chimeric DI RNA was
similar to (76% of) that of WT DI852. As with DI852, DI-SSP RNA
synthesis was dependent on cotransfection of the SV5 L, P, and NP
plasmids (Fig. 6B, lanes 3 to 5, respectively).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 6.
Replication of chimeric DI RNAs containing exchanges of
the SV5 and HPIV2 antigenomic 3' ends. (A) Schematic
representations of chimeric SV5-HPIV2 DI RNAs. Cross-hatched boxes
indicate the termini of the DI RNAs, with white and dark-shaded boxes
representing SV5- and HPIV2-specific sequences, respectively. The
predicted sizes of the DI RNAs are listed, along with the relative
replication level expressed as a percentage (±SD) of that determined
for DI852 (SSS) analyzed in parallel. (B) Replication of the chimeric
DI-SSP RNA requires L, P, and NP proteins. vTF7.3-infected A549 cells
were cotransfected with the SV5 L, P, and NP plasmids along with DNA
encoding either the DI852 genome (lane 1) or the chimera DI-SSP (lane
2). Total intracellular RNA was harvested and analyzed by Northern
blotting using a positive-sense SV5-specific 32P-labeled
riboprobe as described in the legend to Fig. 1. Lanes 3 to 5 are
samples from cells in which the indicated support plasmids were
replaced with pGem control DNA. The arrow indicates the position of
genome-length DI852 RNA. (C) Replication of the SV5-HPIV2 chimeric
antigenome requires sequences contained within HPIV2 CRII. A549 cells
infected with vTF7.3 were transfected with the L, P, and NP plasmids
along with DI852 plasmid (SSS; lane 1), DNA encoding the chimera with
HPIV2 sequences at the 3' terminus (SSP; lane 2) or at the 5' terminus
(PSS; lane 3), or a modified DI-SSP RNA in which HPIV2 CRII sequences
had been deleted (SSPt; lane 4). Total intracellular RNA was harvested
and analyzed by Northern blotting as described above.
|
|
As shown in Fig.
6C for the chimeric DI-PSS antigenome, the terminal 98 HPIV2-specific bases were also capable of functioning
in replication
when positioned at the 5' end of the viral antigenome
(lane 3), and
replication levels typically matched that found
for the WT DI852 DI RNA
(quantitation listed in Fig.
6A). Most
importantly, DI RNA replication
was reduced ~20-fold in the case
of a truncated SV5-HPIV2 chimera
(DI-SSPt [Fig.
6A]) that lacked
the HPIV2 CRII sequences between
antigenome bases 72 and 90 (Fig.
6C, lane 4). Together, these results
indicate that the 3'-terminal
98 bases of the HPIV2 antigenomic RNA can
direct encapsidation
and replication by the SV5 polymerase components,
and RNA replication
is dependent on a region of the antigenome that
includes HPIV2
CRII sequences. The extent of complementarity between
the termini
of DI RNAs has been proposed as a factor that can influence
the
level of replication for vesicular stomatitis virus (VSV)
(
46).
The results presented above suggest that the extent of
terminal
complementarity is not a major factor in SV5 RNA replication,
since the SV5-HPIV2 chimera replicated efficiently despite having
continuous terminal complementarity reduced from 113 (in
DI852)
to 13 bases.
Spacing between CRII and the 3' terminus of the SV5 antigenome is
important for efficient replication of SV5-GL DI RNA analogs.
The
SV5 and HPIV2 antigenomic termini have significant sequence identity in
CRI and CRII but differ considerably in the intervening region,
suggesting that intervening sequences are not critical for replication.
To determine the role of sequences between CRI and CRII in SV5 RNA
replication, a series of DI RNA analogs was constructed to contain
alterations in 3' antigenomic bases 21 to 44, maintaining an overall
6N-length RNA (26).
A
BamHI restriction site was engineered into 3'-terminal
bases 39 to 44 of pDI113-GL-90 DNA to facilitate the construction
of
altered DI analogs. When analyzed in the DI replication assay,
the
113-GL-90B DI RNA analog replicated to levels that closely
matched that
of the WT antigenome (Fig.
7B, lanes 1 and 2), suggesting
that the sequence at this position of the
3'-terminal region was
not critical for SV5 RNA synthesis. Additional
SV5-GL DI RNA analogs
were constructed to contain 5'-to-3' progressive
6-, 12-, and
18-nucleotide deletions of antigenomic RNA bases 38 to 21 (113-GL-90B
6,
-
12, and -
18). RNA synthesis from the
6
antigenome analog was
reduced ~25-fold compared to the 113-GL-90B RNA
(Fig.
7B, lanes
2 and 4; quantitation in Fig.
7A), and replication
products from
the
12 and
18 antigenomes were not detected (Fig.
7B, lanes
5 and 6). In the example shown in Fig.
7B, the film was
purposely
overexposed to illustrate the low level of replication seen
in
the case of the
6 antigenome analog. Likewise, antigenomes
containing
an insertion of 6 or 12 bases between SV5 antigenomic
nucleotides
38 and 39 were defective for replication (Fig.
7B, lanes 7 and
8), and the level of RNA synthesized from these antigenomes was
not
above that detected in control samples from transfected cells
in which
the L protein plasmid had been omitted (lane 3).
View larger version (37K):
[in this window]
[in a new window]
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 7.
Alteration in the spacing between CRII and the 3'
terminus results in SV5 DI RNA analogs that are defective for RNA
replication. (A) Sequences of the 3'-terminal regions of SV5 WT
and mutant antigenomic RNAs. The structure of the 113-GL-90 DI RNA
analog is shown schematically, with the locations of CRI and CRII
sequences indicated by shaded and speckled boxes, respectively. The
nucleotide sequence of the 3'-terminal 71 bases of the SV5 antigenome
is listed below the diagram, with numbers indicating distance from the
3' terminus. The L protein translational stop codon is boxed. Dashes
and underlines in the altered 3' end sequences denote deletions that
created the 6, 12, and 18 antigenomes and nucleotide
replacements that created the 113-GL-90B, -BRe12, -BRe18, and -BRe25
antigenomes, respectively. The locations and sequences of 6 or 12 nonviral bases inserted to create the -B+6 and -B+12 antigenomes are
shown at the bottom. Relative replication levels are expressed as
percentages (±SD) of that determined for the 113-GL-90 (WT) antigenome
analyzed in parallel. (B) Replication of SV5-GL DI RNA analogs
containing alterations in the length and sequence between CRI and CRII.
A549 cells infected with vTF7.3 were transfected with L, P, and NP
plasmids along with DNA encoding one of the indicated SV5-GL DI RNA
analogs. Total intracellular RNA was harvested and analyzed by Northern
blotting with a negative-sense GL-specific riboprobe. Lane 90B-L
represents a sample from cells in which the L plasmid was replaced with
pGem control DNA. The arrow indicates the position of full-length
replication products. Del., deletion mutants; Ins., insertion mutants.
(C) Replication of SV5-GL DI RNA analogs containing a replacement of
sequences between CRI and CRII. Altered SV5-GL antigenomes containing
replacements of bases 14 to 43 were analyzed in the DI replication
assay as described above. Repl., replacement mutants.
|
|
The reduced replication of the above-described deletion/insertion
mutants could result from removal or disruption of an important
cis-acting viral sequence or could reflect a change in
spacing
between the CRI and CRII sequences. To distinguish between
these
possibilities, nonviral nucleotides were inserted in place of
the
bases that had been previously deleted in the case of the
12 and
18 antigenomes, and these replacements restored the overall
length
of the CRI-CRII intervening region (replacement mutants
113-GL-90BRe12
and -Re18). Length-compensating insertions of 12
(Fig.
7C, lane 2) or
18 (lane 3) nonviral bases restored RNA replication
to levels that were
equivalent to that of the parental 113-GL-90B
antigenome (lane
1). Further extending the mutagenesis to include
a replacement of bases
20 to 14 resulted in a ~6-fold reduction
in RNA replication (Fig.
7C,
lane 4), consistent with the proposed
role for CRI sequences in SV5 RNA
synthesis. Together, these data
indicate that the dramatic loss in
replication of deletion mutants
6,
12, and
18 was due to
changes in the spacing between important
antigenomic promoter elements
and was not due to disruption of
a specific sequence requirement per se
in this segment.
| |
DISCUSSION |
Paramyxovirus DI RNAs have proven to be useful models for the
analysis of cis-acting sequences that are necessary for
replication of the viral genome. For the paramyxoviruses that have been
examined to date, all naturally occurring copyback DI RNAs retain ~95
bases or more of terminal complementarity (21, 23, 39, 42), suggesting that this length may be at the lower limit of a functional antigenomic replication promoter. The identification of an important cis-acting sequence located between 3'-terminal bases 73 and
90 of the SV5 antigenome provides a possible explanation for the observed ~90-base minimal extent of terminal complementarity found for paramyxovirus copyback DI RNAs. In support of this
possibility, replication of a foreign gene by paramyxovirus
cis-acting sequences has been reported for SeV
(29), MeV (43), RSV (9), HPIV3 (11, 12), and SV5 (Fig. 3), and in each case, these viral genome analogs retained at least 90 bases from the 3' terminus of the
antigenome.
Sequence alignment identified two conserved regions (CRI and CRII) in
the 3'-terminal 90 bases of the Rubulavirus antigenomic RNA,
and deletions (CRII) or substitutions (CRI) in these sequences reduced
SV5 RNA replication. Several lines of evidence support the proposal
that these two important cis-acting elements are separated
by sequences that function as a spacer region. First, the sequence of
the region located between CRI and CRII is not well conserved between
the rubulaviruses SV5 and HPIV2. Nevertheless, a 3'-terminal RNA
segment containing the HPIV2 CRII-intervening region-CRI sequence was
capable of directing efficient RNA synthesis by the SV5 polymerase
components. These results indicate that while the extent of continuous
3'-terminal complementarity in a rubulavirus genome is not a major
factor determining the efficiency of RNA replication, the presence of
CRI and CRII sequences is essential. Second, the 3'-proximal half of
the intervening region located between SV5 CRI and CRII can be replaced
by nonviral sequences without compromising RNA replication. Finally,
SV5 RNA replication was inhibited by changes of as little as six bases
in the length of intervening sequence between CRII and the 3' terminus,
consistent with the proposal that the relative spacing of CRII and CRI
is an important feature of the SV5 antigenomic promoter. By contrast, a
previous analysis of cis-acting sequences in the le-NP
region of the SeV genome showed that six-base insertions at position 47 or 67 of the genomic RNA did not inhibit SeV DI RNA synthesis, but SeV
genomes containing 12-base insertions in this region were defective for
replication (35). The previous studies and those reported
here are not directly comparable since they involved analyses of the
genomic (for SeV) and antigenomic (SV5) 3' termini for these two
viruses. Nevertheless, it is possible that the requirement during
replication for proper spacing of essential discontinuous cis-acting elements may be a general feature of
paramyxovirus promoter function. Further replacement mutagenesis will
be required to more precisely map the boundaries of these
cis-acting sequences, but these results suggest that the
overall length of the segment located 21 to 44 nucleotides from the 3'
end of the antigenome between CRI and CRII, and not the particular
sequence per se, may be the most important function of this region
during paramyxovirus RNA replication.
The role that SV5 antigenomic CRI and CRII sequences play in RNA
replication is not known. During paramyxovirus replication, RNA
encapsidation by NP is tightly coupled to RNA synthesis (20, 24), and it is possible that one or both of these regions can function as a nucleation site to initiate NP encapsidation on the
nascent RNA. In the case of VSV, previous in vitro work using purified
le RNA (3) or synthetic VSV RNAs (25) indicated that signals for encapsidation are located very near the terminus of
the viral RNA, but similar studies on NP-mediated encapsidation of
paramyxovirus RNAs have not been reported. A deletion of CRII sequences
did not compromise the ability of T7 pol-derived SV5-GL antigenomic RNA
to be encapsidated in vivo, even when the deletion was engineered into
the 5' region of the antigenome. While this result indicates that CRII
sequences are not involved in NP encapsidation of the T7 pol-derived
RNA, it is unclear if this also applies to the steps in NP
encapsidation of nascent RNA synthesized by the viral polymerase.
A possible alternative role for SV5 CRI and CRII in RNA replication is
suggested by the proposed structure of the SeV NC which was previously
derived from electron micrograph images. The SeV NC is thought to exist
as a left-handed helix containing an average of 13 NP molecules per
turn, and each NP molecule appears to bind six nucleotides
(13). Assuming that the SV5 NC structure has these same
properties (8, 10), a hypothetical model for the structure
of the 3' terminus of the SV5 antigenome which is consistent with the
results of the mutational analysis described here can be proposed.
Figure 8 depicts the nucleotide sequence
of the 3'-terminal 113 bases of the SV5 antigenome placed in the
context of ovals, each of which represents one NP molecule binding to
six viral nucleotides, and 13 NP subunits are shown bound to a total of 78 bases (6 times 13) through one turn of a left-handed helix. While
speculative, this postulated structure illustrates the positioning of
CRI (bases 1 to 19) and CRII (bases 73 to 90) along the same face of
the helical NC (Fig. 8). Defects in the replication of antigenomes
containing deletions of bases between CRI and CRII can be corrected by
compensating insertions of non-SV5 sequences, and these results are
consistent with the proposal that RNA synthesis is sensitive to changes
in the spatial alignment of CRI and CRII from their optimal position
along one face of the helical NC.
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 8.
Hypothetical model for the structure of the 3'-terminal
region of the SV5 antigenomic RNA, based on results from the
mutational data presented here and assuming that the SV5 NC has
properties similar to those proposed previously for the SeV NC by
Egelman et al. (13). The nucleotide sequence of the
3'-terminal 113 bases of the SV5 antigenome is listed in the context of
ovals that represent NP monomers. The location of the L protein gene
translational stop codon (UAA) and poly(A) signal are indicated. The
proposed structure aligns SV5 CRI (boldface nucleotides 1 to 19) and
CRII (boldface nucleotides 73 to 90) to the same face of the helical
NC.
|
|
It is possible that the alignment of SV5 antigenomic CRI and CRII
sequences creates a site for polymerase binding. Pelet et al.
(35) have suggested that the SeV polymerase initiates viral RNA synthesis by interacting with at least two separate regions of the
genomic promoter that may be contained within one turn of the helical
NC. Genome replication for many RNA viruses, including brome mosaic
virus (38), flock house virus (2), bacteriophage Q
(5), and yeast L-A virus (15), requires
discontinuous portions of the viral RNA that are located internal to
the 3' terminus. The influenza virus polymerase has been shown to bind to the 5'-terminal end of the viral RNA (45), and both 5'-
and 3'-terminal regions are required for polymerase activity
(17). It has been proposed that the internal or 5'-terminal
sequences in these viral RNAs may function in the assembly or
positioning of the polymerase during the initiation of RNA synthesis,
and the alignment of SV5 CRI and CRII along one face of the helical NC
may serve a similar function.
The model depicted in Fig. 8 has implications for our understanding of
the requirement (6) or the preference (26) for efficient replication of paramyxovirus genomes having an overall 6N
length. The rule of six is thought to operate at the level of
initiation of RNA synthesis (35), and it is proposed that the 3' end of the viral RNA may be recognized by the polymerase complex
most efficiently when it is precisely assembled with NP and no
additional nucleotides protrude from the 3' end of the NC
(6). However, an additional or alternative aspect of the rule of six may be the relative positioning of a particular base within
the hexamer of nucleotides bound by an NP molecule as proposed by Pelet
et al. (35). As such, non-6N-length alterations distal to
the 3'-terminal region of the genomic and antigenomic RNAs may decrease
replication by shifting the relative positions of critical bases within
the context of an NP monomer. As shown in Fig. 8, a conserved 5'-CGR
trinucleotide is found in the 5'-proximal position of each of the SV5
NP-bound hexamers that compose CRII. Preliminary results indicate that
substitutions in these trinucleotides result in SV5 RNA analogs that
are defective in replication (26a), but it is unclear
whether this sequence requirement also involves a position-specific
requirement for these nucleotides within an NP monomer.
Sequences analogous to the SV5 CRII cis-acting element may
be located in the 3'-terminal region of other paramyxovirus antigenomic RNAs. Previous sequence comparisons of several paramyxoviruses, including SeV, HPIV3, and MeV (4), have identified a
conserved sequence located 75 to 95 bases from the end of the RNA (BB
box) that is found in the 3' end of the L protein gene on the
antigenome and in the complement of the 5' end of the NP gene on the
genome. However, the nucleotide sequence of the BB box (4)
is not closely related to the rubulavirus CRII identified here and,
while CRII is clearly essential for RNA synthesis by the SV5
polymerase, the importance of the BB box sequences to RNA replication
for these other paramyxoviruses has not been reported.
The results from the analysis of the SV5 antigenomic promoter reported
here differ from those reported recently for the prototypic rhabdovirus
VSV (22, 33). Using a reverse genetics system, Li and
Pattnaik (22) have identified two regions within the 3'-terminal 45 nucleotides of the VSV antigenome that affect copyback DI RNA replication: an essential region I (the 3'-terminal bases 1 to
24) that is sufficient to direct RNA replication and a nonessential region II (bases 25 to 45) that is proposed to play an
"enhancer-like" function. By contrast, SV5 CRI and CRII are spaced
further apart (within 90 bases) than the corresponding domains in the
VSV antigenome (within 45 bases), and a sequence element within CRII as
well as proper spacing between CRII and the 3' terminus are critical factors that influence SV5 RNA replication. It is proposed that the
presence of this replication-enhancing sequence in the VSV antigenomic
3' end may be responsible for a higher level of RNA synthesized from
the antigenomic than from the genomic promoter (22).
Sequence alignments of the SV5 le and tr' regions revealed that CRI and
CRII are present in the 3' termini of both the SV5 antigenomic and
genomic RNAs. It remains to be determined if differences between the
SV5 antigenomic and genomic CRII sequences are a factor that influences
the level of RNA synthesized from these two promoter regions.
| |
ACKNOWLEDGMENTS |
We thank John Rassa, Si-Yi Chen, Tom Gallagher, and Doug Lyles
for helpful comments on the manuscript.
This work was supported by NIH grant AI34329. Oligonucleotide synthesis
was performed in the DNA Synthesis Core Laboratory of the Cancer Center
of Wake Forest University, supported in part by NIH grant CA-12197.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Wake Forest University Medical Center,
Medical Center Blvd., Winston-Salem, NC 27157-1064. Phone: (910)
716-9083. Fax: (910) 716-9928. E-mail: gparks{at}bgsm.edu.
| |
REFERENCES |
| 1.
|
Ausubel, F.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1995.
.
Current protocols in molecular biology.
John Wiley and Sons, Inc., New York, N.Y.
|
| 2.
|
Ball, L. A., and Y. Li.
1993.
cis-acting requirements for the replication of flock house virus RNA 2.
J. Virol.
67:3544-3551[Abstract/Free Full Text].
|
| 3.
|
Blumberg, B. M.,
C. Giorgi, and D. Kolakofsky.
1983.
N protein of vesicular stomatitis virus selectively encapsidates leader RNA in vitro.
Cell
32:559-567[Medline].
|
| 4.
|
Blumberg, B. M.,
J. Chan, and S. A. Udem.
1991.
Function of paramyxovirus 3' and 5' end sequences. In theory and practice, p. 235-247. In
D. W. Kingsbury (ed.), The paramyxoviruses.
Plenum Press, New York, N.Y.
|
| 5.
|
Brown, D., and L. Gold.
1996.
RNA replication by Q replicase: a working model.
Proc. Natl. Acad. Sci. USA
93:11558-11562[Abstract/Free Full Text].
|
| 6.
|
Calain, P., and L. Roux.
1993.
The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.
J. Virol.
67:4822-4830[Abstract/Free Full Text].
|
| 7.
|
Calain, P., and L. Roux.
1995.
Functional characterisation of the genomic and antigenomic promoters of Sendai virus.
Virology
212:163-173[Medline].
|
| 8.
|
Choppin, P. W., and W. Stoeckenius.
1964.
The morphology of SV5 virus.
Virology
23:195-202[Medline].
|
| 9.
|
Collins, P.,
M. Mink, and D. Stec.
1991.
Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene.
Proc. Natl. Acad. Sci. USA
88:9663-9667[Abstract/Free Full Text].
|
| 10.
|
Compans, R. W., and P. W. Choppin.
1967.
Isolation and properties of the helical nucleocapsid of the parainfluenza virus SV5.
Proc. Natl. Acad. Sci. USA
57:949-956[Free Full Text].
|
| 11.
|
De, B. P., and A. K. Banerjee.
1993.
Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.
Virology
196:344-348[Medline].
|
| 12.
|
Dimmock, K., and P. L. Collins.
1993.
Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3.
J. Virol.
67:2772-2778[Abstract/Free Full Text].
|
| 13.
|
Egelman, E.,
S. Wu,
M. Amrein,
A. Portner, and G. Murti.
1989.
The Sendai virus nucleocapsid exists in at least four different helical states.
J. Virol.
63:2233-2243[Abstract/Free Full Text].
|
| 14.
|
Fuerst, T. R.,
E. G. Niles,
F. W. Studier, and B. Moss.
1986.
Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
Proc. Natl. Acad. Sci. USA
85:8122-8126.
|
| 15.
|
Fujimura, R., and R. B. Wickner.
1992.
Interaction of two cis sites with the RNA replicase of the yeast L-A virus.
J. Biol. Chem.
267:2708-2713[Abstract/Free Full Text].
|
| 16.
|
Grosfeld, H.,
M. G. Hill, and P. L. Collins.
1995.
RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA.
J. Virol.
69:5677-5686[Abstract].
|
| 17.
|
Hagen, M.,
T. D. Y. Chung,
J. A. Butcher, and M. Krystal.
1994.
Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity.
J. Virol.
68:1509-1515[Abstract/Free Full Text].
|
| 18.
|
Hamaguchi, M.,
T. Yoshida,
K. Nishikawa,
H. Naruse, and Y. Nagai.
1983.
Transcriptive complex of Newcastle disease virus.
Virology
128:105-117[Medline].
|
| 19.
|
Kawano, M.,
K. Okamoto,
H. Bando,
K. Kondo,
M. Tsurudome,
H. Komada,
M. Nishio, and Y. Ito.
1991.
Characterizations of the human parinfluenza type 2 virus gene encoding the L protein and the intergenic sequences.
Nucleic Acids Res.
19:2739-2746[Abstract/Free Full Text].
|
| 20.
|
Lamb, R. A., and D. Kolakofsky.
1996.
Paramyxoviridae: the viruses and their replication, p. 1177-1204. In
B. Fields, D. Knipe, and P. Howley (ed.), Fields virology, 3rd ed.
Lippincott-Raven Publishers, Philadelphia, Pa.
|
| 21.
|
Leppert, M.,
L. Kort, and D. Kolakofsky.
1977.
Further characterization of Sendai virus DI-RNAs: a model for their generation.
Cell
12:539-552[Medline].
|
| 22.
|
Li, T., and A. K. Pattnaik.
1997.
Replication signal in the genome of vesicular stomatitis virus and its defective interfering particles: identification of a sequence element that enhances DI RNA replication.
Virology
232:248-259[Medline].
|
| 23.
|
Mottet, G., and L. Roux.
1989.
Budding efficiency of Sendai virus nucleocapsids: influence of size and ends of the RNA.
Virus Res.
14:175-188[Medline].
|
| 24.
|
Moyer, S. A., and S. M. Horikami.
1991.
The role of viral and host cell proteins in paramyxovirus transcription and replication, p. 249-274. In
D. W. Kingsbury (ed.), The paramyxoviruses.
Plenum Press, New York, N.Y.
|
| 25.
|
Moyer, S. A.,
S. Smallwood-Kentro,
A. Haddad, and L. Prevec.
1991.
Assembly and transcription of synthetic vesicular stomatitis virus nucleocapsids.
J. Virol.
65:2170-2178[Abstract/Free Full Text].
|
| 26.
|
Murphy, S. K., and G. D. Parks.
1997.
Genome nucleotide lengths that are divisible by six are not essential but enhance replication of defective interfering RNAs of the paramyxovirus simian virus 5.
Virology
232:145-157[Medline].
|
| 26a.
| Murphy, S. K., and G. D. Parks.
Unpublished data.
|
| 27.
|
Ogawa, M.,
M. Noriko,
M. Tsurudome,
M. Kawano,
H. Matsumura,
S. Kusagawa,
H. Komada,
M. Nishio, and Y. Ito.
1992.
Nucleotide sequence analysis of the simian virus 41 gene encoding the large (L) protein and construction of a phylogenetic tree for the L proteins of paramyxoviruses.
J. Gen. Virol.
73:2743-2750[Abstract/Free Full Text].
|
| 28.
|
Okazaki, K.,
K. Tanabayashi,
K. Takeuchi,
M. Hishiyama,
K. Okazaki, and A. Yamada.
1992.
Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.
Virology
188:926-930[Medline].
|
| 29.
|
Park, K. H.,
T. Huang,
F. F. Correia, and M. Krystal.
1991.
Rescue of a foreign gene by Sendai virus.
Proc. Natl. Acad. Sci. USA
88:5537-5541[Abstract/Free Full Text].
|
| 30.
|
Parks, G. D.
1994.
Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P.
J. Virol.
68:4862-4872[Abstract/Free Full Text].
|
| 31.
|
Parks, G. D.,
C. D. Ward, and R. A. Lamb.
1992.
Molecular cloning of the NP and L genes of simian virus 5: identification of highly conserved domains in paramyxovirus NP and L proteins.
Virus Res.
22:259-279[Medline].
|
| 32.
|
Pattnaik, A. K.,
L. A. Ball,
A. W. Legrone, and G. W. Wertz.
1992.
Infectious defective interfering particles of VSV from transcripts of a cDNA clone.
Cell
69:1011-1020[Medline].
|
| 33.
|
Pattnaik, A. K.,
L. A. Ball,
A. LeGrone, and G. W. Wertz.
1995.
The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles.
Virology
206:760-764[Medline].
|
| 34.
|
Pattnaik, A. K., and G. W. Wertz.
1990.
Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs.
J. Virol.
64:2948-2957[Abstract/Free Full Text].
|
| 35.
|
Pelet, T.,
C. Delenda,
O. Gubbay,
D. Garcin, and D. Kolakofsky.
1996.
Partial characterization of a Sendai virus replication promoter and the rule of six.
Virology
224:405-414[Medline].
|
| 36.
|
Perrault, J.
1981.
Origin and replication of defective interfering particles.
Curr. Top. Microbiol. Immunol.
93:151-207[Medline].
|
| 37.
|
Perrotta, A. T., and M. D. Been.
1991.
A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA.
Nature (London)
350:434-436[Medline].
|
| 38.
|
Quadt, R.,
M. Ishikawa,
M. Janda, and P. Ahlquist.
1995.
Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.
Proc. Natl. Acad. Sci. USA
92:4892-4896[Abstract/Free Full Text].
|
| 39.
|
Re, G.
1991.
Deletion mutants of paramyxoviruses, p. 275-298. In
D. W. Kingsbury (ed.), The paramyxoviruses.
Plenum Press, New York, N.Y.
|
| 40.
|
Rose, J. K.,
L. Buonocore, and M. A. Whitt.
1991.
A new cationic liposome reagent mediating nearly quantitative transfection of animal cells.
BioTechniques
10:520-525.
[Medline] |
| 41.
|
Samal, S., and P. Collins.
1996.
RNA replication by a respiratory syncytial virus analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end.
J. Virol.
70:5075-5082[Abstract/Free Full Text].
|
| 42.
|
Sidhu, M.,
J. Crowley,
A. Lowenthal,
D. Karcher,
J. Menonna,
S. Cook,
S. Udem, and P. Dowling.
1994.
Defective measles virus in human subacute sclerosing panencephalitis brain.
Virology
202:631-641[Medline].
|
| 43.
|
Sidhu, M.,
J. Chan,
K. Kaelin,
P. Spielhofer,
F. Radecke,
H. Schneider,
M. Masurekar,
P. Dowling,
M. Billeter, and S. Udem.
1995.
Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene.
Virology
208:800-807[Medline].
|
| 44.
|
Tapparel, C., and L. Roux.
1996.
The efficiency of Sendai virus genome replication: the importance of the RNA primary sequence independent of terminal complementarity.
Virology
225:163-171[Medline].
|
| 45.
|
Tiley, L. S.,
M. Hagen,
J. T. Matthews, and M. Krystal.
1994.
Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.
J. Virol.
68:5108-5116[Abstract/Free Full Text].
|
| 46.
|
Wertz, G.,
S. Whelan,
A. LeGrone, and L. Ball.
1994.
Extent of terminal complementarity modulates the balance between transcription and replication of vesicular stomatitis virus RNA.
Proc. Natl. Acad. Sci. USA
91:8587-8591[Abstract/Free Full Text].
|
J Virol, January 1998, p. 10-19, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Manuse, M. J., Parks, G. D.
(2009). Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing. J. Virol.
83: 9057-9067
[Abstract]
[Full Text]
-
Walpita, P., Peters, C. J.
(2007). Cis-acting elements in the antigenomic promoter of Nipah virus. J. Gen. Virol.
88: 2542-2551
[Abstract]
[Full Text]
-
Cowton, V. M., McGivern, D. R., Fearns, R.
(2006). Unravelling the complexities of respiratory syncytial virus RNA synthesis. J. Gen. Virol.
87: 1805-1821
[Abstract]
[Full Text]
-
Hoffman, M. A., Thorson, L. M., Vickman, J. E., Anderson, J. S., May, N. A., Schweitzer, M. N.
(2006). Roles of human parainfluenza virus type 3 bases 13 to 78 in replication and transcription: identification of an additional replication promoter element and evidence for internal transcription initiation.. J. Virol.
80: 5388-5396
[Abstract]
[Full Text]
-
Cordey, S., Roux, L.
(2006). Transcribing paramyxovirus RNA polymerase engages the template at its 3' extremity.. J. Gen. Virol.
87: 665-672
[Abstract]
[Full Text]
-
Cowton, V. M., Fearns, R.
(2005). Evidence that the Respiratory Syncytial Virus Polymerase Is Recruited to Nucleotides 1 to 11 at the 3' End of the Nucleocapsid and Can Scan To Access Internal Signals. J. Virol.
79: 11311-11322
[Abstract]
[Full Text]
-
Kolakofsky, D., Roux, L., Garcin, D., Ruigrok, R. W. H.
(2005). Paramyxovirus mRNA editing, the 'rule of six' and error catastrophe: a hypothesis. J. Gen. Virol.
86: 1869-1877
[Abstract]
[Full Text]
-
Vulliemoz, D., Cordey, S., Mottet-Osman, G., Roux, L.
(2005). Nature of a paramyxovirus replication promoter influences a nearby transcription signal. J. Gen. Virol.
86: 171-180
[Abstract]
[Full Text]
-
Le Mercier, P., Garcin, D., Garcia, E., Kolakofsky, D.
(2003). Competition between the Sendai Virus N mRNA Start Site and the Genome 3'-End Promoter for Viral RNA Polymerase. J. Virol.
77: 9147-9155
[Abstract]
[Full Text]
-
Neumann, G., Whitt, M. A., Kawaoka, Y.
(2002). A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?. J. Gen. Virol.
83: 2635-2662
[Abstract]
[Full Text]
-
Vulliemoz, D., Roux, L.
(2002). Given the Opportunity, the Sendai Virus RNA-Dependent RNA Polymerase Could as Well Enter Its Template Internally. J. Virol.
76: 7987-7995
[Abstract]
[Full Text]
-
Le Mercier, P., Garcin, D., Hausmann, S., Kolakofsky, D.
(2002). Ambisense Sendai Viruses Are Inherently Unstable but Are Useful To Study Viral RNA Synthesis. J. Virol.
76: 5492-5502
[Abstract]
[Full Text]
-
Mioulet, V., Barrett, T., Baron, M. D.
(2001). Scanning mutagenesis identifies critical residues in the rinderpest virus genome promoter. J. Gen. Virol.
82: 2905-2911
[Abstract]
[Full Text]
-
Keller, M. A., Murphy, S. K., Parks, G. D.
(2001). RNA Replication from the Simian Virus 5 Antigenomic Promoter Requires Three Sequence-Dependent Elements Separated by Sequence-Independent Spacer Regions. J. Virol.
75: 3993-3998
[Abstract]
[Full Text]
-
Coronel, E. C., Takimoto, T., Murti, K. G., Varich, N., Portner, A.
(2001). Nucleocapsid Incorporation into Parainfluenza Virus Is Regulated by Specific Interaction with Matrix Protein. J. Virol.
75: 1117-1123
[Abstract]
[Full Text]
-
Stillman, E. A., Whitt, M. A.
(1999). Transcript Initiation and 5'-End Modifications Are Separable Events during Vesicular Stomatitis Virus Transcription. J. Virol.
73: 7199-7209
[Abstract]
[Full Text]
-
Peeters, B. P. H., de Leeuw, O. S., Koch, G., Gielkens, A. L. J.
(1999). Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence. J. Virol.
73: 5001-5009
[Abstract]
[Full Text]
-
Finke, S., Conzelmann, K.-K.
(1999). Virus Promoters Determine Interference by Defective RNAs: Selective Amplification of Mini-RNA Vectors and Rescue from cDNA by a 3' Copy-Back Ambisense Rabies Virus. J. Virol.
73: 3818-3825
[Abstract]
[Full Text]
-
Whelan, S. P. J., Wertz, G. W.
(1999). Regulation of RNA Synthesis by the Genomic Termini of Vesicular Stomatitis Virus: Identification of Distinct Sequences Essential for Transcription but Not Replication. J. Virol.
73: 297-306
[Abstract]
[Full Text]
-
Fearns, R., Collins, P. L.
(1999). Model for Polymerase Access to the Overlapped L Gene of Respiratory Syncytial Virus. J. Virol.
73: 388-397
[Abstract]
[Full Text]
-
Murphy, S. K., Parks, G. D.
(1999). RNA Replication for the Paramyxovirus Simian Virus 5 Requires an Internal Repeated (CGNNNN) Sequence Motif. J. Virol.
73: 805-809
[Abstract]
[Full Text]
Top
Abstract
Introduction
