-
. 'I'.••
Acknowledgements
Plasmid pYT1 04V was graciously provided to us by Dr. Peter
Tattersall.
The research which is described in this paper was
carried out during the summer of 1989 under the supervision of Dr.
Arun Srivastava of the Indiana University School of Medicine,
Department of Microbiology and Immunology.
I would like to
express my gratitude to Sandy Jackson and Piruz Naherini for their
helpfulness during the course of this project.
I would also like to
express my thanks to the Honors College and Dr. J.R. Garcia for
giving me my start in scientific research during the year of my
undergraduate fellowship
1988 - 89.
In vitro transcription and translation of the human
pathogenic parvovirus Bl9 nonstructural (NSl) gene.
In vitro transcription and translation of the human
pathogenic parvovirus Bl9 nonstructural (NSl) gene.
An Honors Thesis
by
Mike Woody
Thesis Director
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V
V !: \ / '<L vC-c=~
Ball State University
Muncie, Indiana
MayJ990
Expected Date of Graduation
MayJ990
·.
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(;..'
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Acknowledgements
Plasmid pYT104V was graciously provided to us by Dr. Peter
Tattersall.
The research which is described in this paper was
carried out during the summer of 1989 under the supervision of Dr.
Arun Srivastava of the Indiana University School of Medicine,
Department of Microbiology and Immunology.
I would like to
express my gratitude to Sandy Jackson and Piruz Naherini for their
helpfulness during the course of this project.
I would also like to
express my thanks to the Honors College and Dr. J.R. Garcia for
giving me my start in scientific research during the year of my
undergraduate fellowship
-
1988 - 89.
1
M. Woody
Abstract The human parvovirus B19 contains inverted terminal
repeats (ITRs) which form stable hairpin structures. Although ITRs
have not been shown to be transcribed, recent studies indicate
that they function as enhancers.
To define the role of ITRs in
transcription, we subcloned the B19 non-structural (NS1) gene,
with or without the ITR sequence upstream of the NS1 gene, into a
prokaryotic vector pSP65.
Plasmid pWS602 contained the ITR
sequence whereas this sequence was deleted in plasmid pWS603.
Both plasm ids were transcribed in. vitro using the SP6 RNA
transcription system.
Equivalent amounts of in vitro synthesized
mRNAs were translated in vitro using the rabbit reticulocyte lysate
system.
Whereas mRNA transcribed from pWS603 yielded two NS1
proteins of mol. wts. 78 kD and 68 kD respectively, no translation
of NS1 proteins occurred with mRNA transcribed from pWS602.
These studies document that the presence of a hairpin structure
upstream of the NS1 gene has no effect on the efficiency of
transcription but effectively inhibits translation of the NS1 gene
products ill vitro.
These results are thus consistent with the
observed lack of expression of ITRs which, in turn, would
negatively affect the production of viral gene products in vi.v.Q..
-
2
M. Woody
Parvoviruses are among the smallest of the DNA-containing viruses
that infect a wide variety of vertebrates. Human pathogenic
parvovirus 819 has been shown to be the etiologic agent of red cell
aplasia in patients with hemolytic anemia (Saarinen, Chorba and
Tattersall, 1986) as well as the causative agent in some cases of
hydrops fetal is (8rown et al., 1984). 819 has been demonstrated to
cause the childhood disease, erythema infectiosum commonly
called the "fifth disease" (Anderso n et aI., 1984). 819 is also linked
to transient post-infection arthropathy in adults (White et aI.,
1985), and has recently been isolated from synovial fluid (Dijkmans
et aI., 1988).
The 819 viral genome is single stranded DNA of approximately
5.4 kb in length (Shade et al., 1986).
819 is an autonomously
replicating virus which requires mitotically active cells for its
propagation (Ozawa, Kurtzman and Young, 1986). Thus far, 819 has
only been shown to replicate in human erythroid progenitor cells in
the bone marrow (Young et aI., 1984) and fetal liver (Yaegashi et
aI., 1989) . The right half of the 819 viral genome codes for at least
two capsid proteins of 84 KD and 58 KD, (Cotmore et aI., 1986)
while the left half codes for a 77 KD nonstructural protein (Ozawa
et aI., 1988).
-
80th the 3' and 5' termini of the 819 genome are
3
M. Woody
flanked by regions of G-C rich inverted terminal repeats (ITRs)
which form a stable hairpin-like secondary structure (Shade et aI.,
1986). and fuction as primers in viral DNA replication (Tattersall and
Cotmore, 1984).
The ITRs account for nearly 13% of the viral DNA,
yet have not been shown to be transcribed during cases of B19
infection. To further define the role of the B19 ITRs in transcription
and translation, we deleted the right half of the B19 genome and
subcloned the B19 non-structural gene (NS1) with or without the
ITR sequence upstream of the initiation site of transcription of the
viral NS1 gene, into a prokaryotic vector pSP65.
Transcription of
B19-DNA within the cloning vector pSP65 was driven from a SP6
promoter upstream of the viral DNA.
Two constructs were made;
the plasmid designated pWS602 contained the NS1
downstream from the ITR sequence.
gene
pWS603 contained the NS1
gene, but the upstream ITR sequence was deleted.
In this report,
we document that the presence of a hairpin-like structure
upstream of the viral mRNA coding for the NS1 gene product
negatively
affects
its
translation
Materials
in. vi t ro.
and
Methods
Plasmid Constructions. Plasmid pYT104V contains the fulllength genome of the human pathogenic parvovirus B19 with the
4
M. Woody
exception of some deletions in parts of the inverted termini at both
the 5' and 3' ends of the genome (Shade et aI., 1986). The viral DNA
has been cloned into the polylinker region of the pSP65 cloning
vector, approximately 22 bases downstream from a SP6
bacteriophage promoter.
Putative sense mRNA is the product of in
vitro transcription from the SP6 promoter.
In order to isolate the
nonstructural gene we removed the right half of the B19 genome
which codes for the capsid proteins by digestion with Pvu II.
We
isolated a 5.84 kb fragment on a 1 % agarose gel and religated to
generate the plasmid pWS602. pWS602 contained B19 DNA bases 13050. A TATA sequence at B 19 nt 319 is believed to serve as the left
had promoter for the NS1 gene in the plasmid pYT104V as well as in
pWS602 (Shade et aI., 1986).
The initiator codon (ATG) for the B19
NS1 gene occurs at nt 436 and is followed by an open reading frame
ending with TGA at nt 2445, yielding an expected protein sequence
of 671 amino acids (Shade et aI., 1986). Bases 1-436 represent the
5' noncoding region which contains the viral ITR.
To prepare
plasmid pWS603 we linearized pWS602 with EcoR1 which cuts once
within the polylinker region between the SP6 promoter and viral
ITR, and then digested completely with Hpa I which cleaves B19 DNA
at base 424 and 1488.
-
A small fragment containing the hairpin
5
M. Woody
-
region ITR DNA was discarded.
An intermediate fragment nt 424-
1488 containing the left half of the NS1 gene was religated to the
vector whose ends were repaired with klenow fragment to
generate pWS603.
Correct orientation was tested by restriction
digest with Hpa I and Xba I.
The general overall strategy of
construction of pWS602 and pWS603 is depicted in Figure - 1.
In vitro transcription of B19 NS1 gene in plasm ids pWS602
and pWS603 was done by using a SP67T7 transcription kit purchased
from Boehringer Mannheim (Indianapolis).
B19 viral DNA inserted
into a pSP65 cloning vector was linearized with Pvu II and
transcribed as described in the manufacturers directions under
"cold" transcription.
The reaction was allowed to proceed at 37
deg. C for 60 minutes at which time 1 microliter of DNAse was
added to react for ten additional minutes to stop the reaction.
One
microliter of the transcription mixture was then analyzed on a 1%
agarose mini-gel while the rest of the mRNA was frozen at -80 deg.
C.
In vitro translation.
Equivalent amounts of in. vitro
synthesized RNA transcripts were translated in a reticulocyte
system (Promega) as described by the manufacturer, and the
translation products were analyzed by 12% polyacrylamide - SDS
-
6
M. Woody
gel electrophoresis.
Samples were electrophoresed at 15 mA for
two hours followed by 30 mA for an additional 7 hours. The
in. vitro
translation reaction was allowed to proceed at 30 deg. C for one
hour using 1 x, 2x or 4x concentrations of RNA synthesized in vitro
from pWS602 or pWS603 templates.
The gel was fixed, stained,
dried, and autoradiographed at -80 deg. C for 48 hours.
Results
Analysis of RNA synthesized following
in. vitro transcription of
plasmids pWS602 and pWS603 on 1 % agarose after removal of
template DNA by DNAse, confirmed the presence of mRNA
transcripts (data not shown).
Equivalent amounts of mRNA
transcripts were subsequently translated
in. vitro.
The results of
12% SDS-polyacrylamide gel electrophoresis are shown in Figure 2. RNA synthesized from plasmid pWS603 yielded two NS1 proteins
of 78 KD and 68 KD as expected when translated
in. vitro.
No
translation product was observed for mRNA transcribed from
pWS602, whose 5' noncoding region DNA contained the inverted
terminal repeats.
Lanes 3, 4, and 5 contained the translational
product from 1 x, 2x, and 4x concentration of mRNA from pWS602.
No specific bands were present in these lanes indicating that
-
7
M. Woody
-
translation of the viral NS1 gene did not occur.
Lanes 7, 8, and 9
contained
1 x,
the
translation
product
concentrations of mRNA from pWS603.
from
2x
and
4x
These lanes show the
expected 78 KD and 68 KD proteins which increase in band intensity
with increasing RNA concentrations.
The band which is clearly
present around 50 KD is an endogenous protein present even in no
RNA control assays (data not shown).
Several lower mol. wt.
proteins can be visualized on the gel in lanes 3, 4, 5, 7, 8, and 9
which are believed to be the result of internal initiation from
downstream AUGs.
characteristic
The formation of these small proteins is
of protein
synthesis in vi t ro but not in ld.v..Q..
Discussion
8y forcing the transcription of the 819 left end terminal ITR in
vitro, we have essentially introduced additional 5' noncoding region
mRNA upstream of the initiation site of the 819 nonstructural gene.
Due to several physical properties of the ITR, such as its
complementary base pairing, high G-C content, and its close
proximity to the 5' cap, the noncoding region would be expected to
form stable secondary structure upstream from the initiation
codon of the nonstructural gene.
Assuming a scanning model for
8
M. Woody
the mechanism of translation (Kozak, 1978), the resulting
secondary structure would be expected to down regulate
translation of the NS1 gene.
The scanning model postulates that a
40s ribosomal subunit binds initially at the 5' end of the mRNA at
the 5' cap region and immigrates along the RNA until it reaches the
first AUG triplet (Kozak, 1978, 1980, 1989b). An updated version of
the scanning model allows for initiation of sites downstream from
the first AUG in some mRNAs providing the first AUG does not occur
within an "optimal" context (Kozak, 1987).
The scanning model
predicts that any secondary structure upstream from the AUG
initiator would down regulate translation by inhibiting the linear
motion of the 40s ribosomal subunits from their entrance at the 5'
cap to the initiator codon (Kozak, 1986, 1989a).
Furthermore, this
inhibition might be expected to increase as the secondary structure
was in closer proximity to the 5' cap since this structure is thought
to be the site of ribosomal entry onto mRNA (Kozak, 1986, 1989a).
The 819 ITR which we transcribed in vitro from 819 DNA in pWS602
formed stable hairpin structure
-100 Kcal/mole, approximately
22 nucleotides upstream from the 5' cap.
Kozak has previously
demonstrated that an artificially introduced hairpin
G
-50
Kcal/mole at the midpoint of the 5' noncoding sequence inhibited
-
9
M. Woody
translation of preproinsulin by 85 - 95%.
structure however,
G
A less stable hairpin
-30 Kcal/mole did not inhibit translation
of the gene when it was inserted at an equivalent position in the
genome (Kozak, 1986).
Similar findings were reported by Pelletier
and Sonenberg who observed decreased translational efficiency of
the thymidine kinase gene of herpes simplex virus 1 which was
mutated by the addition of hairpin forming oligodeoxynucleotide
linkers into the 5' untranslated portion of the mRNA (1985).
There
are several reports that nontranslated leader sequences on viral
mRNAs can be deleted without deleterious effects on translation.
.-
Spindler and Berk observed that complete deletion of wild type 5'
untranslated sequence mRNAs did not significantly affect the rate
of translation of adenovirus mRNAs (1984).
Other studies which
have yielded similar findings regarding the lack of "enhancer"
ability in the 5' noncoding region mRNA were done by Bendig et aI.,
1979, and Villarreal et al., 1978.
It has also been previously
demonstrated that nontranslated 5' regions of mRNA may down
regulate gene expression (Darveau et al., 1985).
Darveau
demonstrated that the translational efficiency of murine C-myc
could be increased 10 fold when the mRNA in the 5' noncoding
region was decreased from 448 nucleotides to 83 nucleotides ,
10
M. Woody
possibly due to the fact that regions of secondary structure were
deleted in the smaller transcript (Darveau, Pelletier, Sonenberg,
1985).
In our study, the formation of a stable hairpin formed by a
transcribed ITR near the 5' cap in the noncoding region of the mRNA
abolished translation of the downstream NS1 gene in the human
pathogenic parvovirus 819.
We believe that the stable secondary
structure formed by the 5' noncoding region stalled the ribosomes
upstream of the initiator codon of the NS1 gene, thereby blocking
initiation of translation.
Our findings are consistent with the
scanning model of translation since deletion of the 5' noncoding
region containing the ITR resulted in the ability of the NS1 gene to
be translated.
The possibility exists that the secondary structure
formed by the ITR at the 5' end of the mRNA may have prevented
primary ribosomal binding to the mRNA since the hairpin occurred in
such close proximity to the 5' cap.
Our data is consistent with the
observed lack of transcription of viral ITRs during 819 infections
in.
Y.iY.Q. which, in turn, would negatively affect the production of the
viral proteins within the infected cells by interfering with
translation of viral genes located downstream from the region of
stable
secondary
structure.
CONSTRUCTtON OF PLASa.JS pWSI02 ANO pWS803
+~
....
(lncorreet on.allonl
~~~.J~!
FIGURE 1. General strategy for the construction of p1asmids
pWS602 and pWS603.
-
pWS602
pWS603
kD
o
116.2
9aS
FIGURE 2. A very stable hairpin structure (ITR) close
to the 5' cap inhibits translation.
pWS602 RNA transcripts
contain secondary structure that eliminates translation of
the B19 NSI gene in vitro. pWS603 RNA in which the ITR :
was deleted prior to transcription can be translated to yield
the expected 68kD and 78kD proteins in vitro. The black
arrows indicate the position of the expected NSl gene products.
11
M. Woody
Cited
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