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J Virol, June 1998, p. 4783-4788, Vol. 72, No. 6
Fox Chase Cancer Center, Philadelphia,
Pennsylvania 19111-2497,1 and
Harvard
Medical School, Boston, Massachusetts 021152
Received 11 December 1997/Accepted 3 March 1998
The small, 195-amino-acid form of the hepatitis delta virus (HDV)
antigen ( Human hepatitis delta virus (HDV) is
a subviral satellite of hepatitis B virus, on which it is dependent for
its envelope proteins (16). HDV particles contain a
ribonucleoprotein (RNP) core composed of the circular 1.7-kb RNA genome
and multiple copies of the delta antigen ( Both The nature of the essential role(s) of While much valuable information can be gained about HDV genome
replication by the use of cDNA transfections, there are obvious inadequacies when it comes to studying the early events of RNA-directed RNA synthesis. We and others have attempted transfections with HDV RNAs
(10, 12). Consistent with the cDNA result, transfection of
cells with HDV RNA isolated from virions or genomic RNA transcribed in
vitro does not lead to initiation of genome replication; however, if
the cells already contain In the present study, we have used an RNP transfection approach to
investigate the early role(s) of Expression plasmids.
pR5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Initiation of Hepatitis Delta Virus Genome
Replication
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Ag-S) is essential for genome replication, i.e., for the
transcription, processing, and accumulation of HDV RNAs. To better
understand this requirement, we used purified recombinant Ag-S and
HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs
into human cells, we detected HDV genome replication, as assayed by
Northern analysis or immunofluorescence microscopy. Our interpretation
is that the input Ag-S is necessary for the RNA to undergo limited
amounts of RNA-directed RNA synthesis, RNA processing, and mRNA
formation, leading to de novo translation of Ag-S. It is this second
source of Ag-S which then goes on to support genome replication.
This assay made it possible to manipulate in vitro the composition of
the RNP and then test in vivo the ability of the complex to initiate
RNA-directed RNA synthesis and go on to achieve genome replication. For
example, both genomic and antigenomic linear RNAs were acceptable.
Substitution for Ag-S with truncated or modified forms of the Ag,
and even with HIV nucleocapsid protein and polylysine, was
unacceptable; the exception was a form of Ag-S with six histidines
added at the C terminus. We expect that further in vitro modifications
of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Ag), the only HDV-encoded
protein (25). There are two main forms of the Ag. The
small 195-amino-acid (aa) form, Ag-S, is essential for genome
replication (14). However, during replication,
posttranscriptional RNA editing at a site corresponding to the
termination codon for Ag-S allows the synthesis of an extra 19 aa to
make the larger, 214-aa form, Ag-L (19).
Ag-S and Ag-L form a ribonucleoprotein complex with HDV RNA,
and some of the functions of these proteins are probably mediated
following incorporation into such complexes. Both proteins are found
within the RNP core of mature virus (25).
Ag-L appears to have two roles late in virus replication. First,
Ag-L can act as a dominant-negative inhibitor of genome replication
(5). Thus, its natural appearance via editing could facilitate a suppression of RNA accumulation. Second, it is essential for particle assembly (4). There is evidence that this
assembly is dependent on farnesylation of the cysteine 4 aa from the
end of the 19-aa C-terminal extension (9).
Ag-S in virus replication is
less clear. Attempts to achieve genome replication by transfection of
cells with viral cDNAs have been successful, but only when the cDNA
encodes a functional Ag-S protein (14). It is not known
whether Ag-S has some cotranscriptional role in viral RNA synthesis
in vivo. Transcription of HDV RNA in nuclear extracts was achieved by
the host RNA polymerase II in the absence of Ag-S (8,
20); however, further studies are needed to confirm this and to
directly test the effects of addition of Ag-S. It is clear that in
vivo, Ag-S does have other roles. It stabilizes HDV RNA circles
(18), and it facilitates ribozyme cleavage (13). Also, since Ag-S has both a nuclear localization signal and
RNA-binding activity that is HDV RNA specific (12), it
probably functions in both the initial transport of HDV RNP to the
nucleus and the maintenance in this location during RNA-directed RNA
synthesis.
Ag-S, then replication occurs
(10). This requirement for Ag-S can also be met if cells
are transfected with virions or viral RNP; under these conditions, the
RNA presumably enters the cells already complexed with a mixture of
Ag-S and Ag-L (1).
Ag-S in events leading to
RNA-directed RNA synthesis. Our strategy was to assemble this RNP in
vitro, using recombinant Ag-S and RNAs as synthesized in vitro. We
found that after cells were transfected with such RNP, we could detect
HDV genome replication. Application of this approach has allowed us to
begin to define some of the protein and RNA requirements for the early
events of HDV genome replication.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
V5 was constructed for the
high-level expression of Ag-S in Escherichia coli. The
protein sequence of the American strain with the Ag-S (GenBank
accession no. M28267) was back-translated with the program
BACKTRANSLATE. (This program was from the Wisconsin Package, version
9.0 [Genetics Computer Group, Madison, Wis.], with E. coli
codon frequencies obtained from
gopher://weeds.mgh.harvard.edu:70/Oftp%3Aweeds.mgh.harvard.eduA@/pub/codon/eco.cod.) With the sequence obtained as shown in Fig.
1, the plasmid pR5V5 was constructed
by a two-step PCR method, as described previously (3), with
the exception that Vent polymerase (New England BioLabs) was used
instead of Taq polymerase. Eight overlapping synthetic primers were synthesized (Fig. 1). Changes in the back-translated sequence were made so that the overlaps of the PCR primers would have
approximately the same melting temperature. Primers were electrophoresed into a 10% sequencing gel, visualized by UV shadowing, and excised from the gel. The primers were then purified with a Waters
Sep-Pak column.
View larger version (78K):
[in a new window]
FIG. 1.
Sequence of synthetic gene for optimized expression of
Ag-S in E. coli. The underlined sequences correspond to
the eight primers used in the first round of PCR. The primers used in
the second round are indicated with a dotted underline. The amino acid
sequence is shown above the DNA sequence by the one-letter amino acid
code. The restriction sites used in cloning are shown in italics.
Ag-S was
subcloned into expression vector pRSETb (Invitrogen). The sequence of
the resultant plasmid, pR5V5, was verified by dye termination
sequencing.
Plasmids pDL444 and pDL445 were constructed to express Ag-S and
Ag-L, respectively, under the control of the simian virus 40 late
promoter (17).
The plasmids described below were constructed to express proteins with
a tag of six histidine residues. The following convention was adopted
to indicate whether the tag was N or C terminal: His6Ag for an N-terminal tag and AgHis6 for a C-terminal tag.
Plasmid pMB001 was constructed to express Ag-SHis6 in
E. coli. Three DNA fragments were ligated: fragment 1 was
cut from pDL670 (17) with AlwnI and
NcoI, fragment 2 was cut from pGemex2 (Promega) with
AlwnI and SalI, and fragment 3 was amplified by PCR from pDL444 and then cut with NcoI and SalI.
pTW203, which expresses Ag-SHis6 via the cytomegalovirus
immediate-early promoter, was constructed as follows. The cDNA of Ag-S was amplified by PCR with primers that added six C-terminal histidines and then was cloned into vector pcDNA3.1/HisA (Invitrogen), which had been modified to allow initiation at the natural start of
Ag-S.
pVB102 was constructed to express His6Ag-L under control
of the immediate-early CMV promoter. The Ag-L open reading frame was
amplified by PCR, cut with BamHI and XbaI, and
ligated to the BamHI and XbaI sites of
pcDNA3.1/His6A (Invitrogen). pVB103 was similarly constructed to
express His6Ag-L, with cysteine 211 changed to alanine.
p1-84His6 was made to express a truncated form of Ag.
It was made by cutting pR5V5 with ApaI. Linkers were
added to incorporate a histidine tag after residue 84, followed by a
stop codon and an NcoI site. NcoI digestion was
used to delete the remainder of the Ag-S gene, and the plasmid was
religated.
pHIV-1NC, made for the expression in E. coli of the 71-aa
form of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC), with the sequence Met-(His)6 added at the N-terminal end,
was obtained from Patrick Brown (28).
pSVL(D2M) (14) expresses a mutant dimer of the HDV genome
with a 2-nucleotide deletion at position 1434 to 1435 which eliminates the ability to synthesize Ag.
The various forms of Ag expressed by the plasmids described above
are summarized in Fig. 2.
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Protein purification.
Recombinant Ag-S was expressed and
purified as follows. Plasmid pR5V5 was transformed into
BL21(DE3)pLysS cells (Novagen). A single colony was used to inoculate a
100-ml overnight culture. Ten milliliters of this overnight culture was
used to inoculate a 1-liter culture. At an optical density of between
0.4 and 0.6, the cells were induced with 3 ml of 100 mM IPTG
(isopropyl-
-D-thiogalactopyranoside). Cell growth was
continued for 3 h, and then cells were pelleted at 5,000 × g for 10 min. The cells were resuspended in 15 ml of 50 mM
HEPES (pH 7.5)-250 mM NaCl-1 mM MgCl2 and stored at
20°C until needed.
Ag-S
were applied to a Superdex S-200 column (Pharmacia) equilibrated with 50 mM HEPES (pH 7.5), 500 mM NaCl, and 5% glycerol. The Ag-S obtained was >85% pure as judged by Coomassie blue staining of a
sodium dodecyl sulfate gel.
Proteins with a histidine tag were purified as follows. Proteins
expressed in E. coli were affinity purified with a Talon column according to the recommendations of the manufacturer (Clontech). Proteins expressed in mammalian cells were purified by the Invitrogen Xpress System. In both cases, the fractions containing the purified protein were identified by sodium dodecyl sulfate gel electrophoresis, pooled, dialyzed, and concentrated.
Other proteins.
The peptide Ag12-60Y was synthesized and
purified as described by Rozzelle et al. (24).
Poly-D-lysine (peak molecular mass, 513 kDa) was obtained
from Sigma.
RNA transcription. For RNP reconstitution experiments, we used genomic or antigenomic HDV RNA synthesized in vitro. A 1.2× unit-length cDNA was put under control of T7 promoter and terminator sequences in a modified pGEM4Z vector (Promega). The resulting plasmids, pTW101 for genomic RNA and pTW114 for antigenomic RNA, were used as the templates for RNA transcription (18).
RNP assembly. Purified RNA and protein were combined in 10 µl of buffer (5 mM HEPES [pH 7.5], 50 mM NaCl, 0.5% glycerol) and incubated for 5 min at room temperature.
Nondenaturing gel electrophoresis of RNP. Horizontal gels of 1.0% agarose were cast in Tris-borate-EDTA (TBE) buffer (90 mM boric acid, 10 mM Tris-base [pH 8.0], 1 mM EDTA). RNP samples were mixed with gel loading buffer (1× TBE, 30% glycerol, 0.2% bromophenol blue), and electrophoresis was performed at 120 V until the bromophenol blue dye front had migrated 8 cm.
Transfection.
Plastic 16-mm-diameter tissue culture wells
(Costar) were seeded with approximately 0.1 × 106
Huh7 cells (21). For transfections with assembled RNP, 0.25 to 900 ng of Ag-S and 500 ng of genomic HDV RNA in 125 µl of Opti-MEM were combined with 2.7 µl of lipofectamine (2 mg/ml) in 125 µl of Opti-MEM, incubated for 30 min at room temperature, and applied
to cells that had been washed with Opti-MEM (11). In control
transfections, either Ag-S or HDV RNA was omitted. For cDNA
transfections, 500 ng of plasmid DNA was used. At 5 h after
transfection, the transfection mixture was changed to Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. At 4 days after transfection, cells were reseeded into a 30-mm-diameter dish
containing a glass coverslip; at 8 days, the cells were examined by
immunofluorescence microscopy. We picked 8 days for three reasons. (i)
We needed to avoid detection of the transfected Ag-S. (ii) In the
immunofluorescence assays, 8 days corresponded to the peak signal for a
cell undergoing RNP-initiated replication. (iii) At 8 days, we could
readily detect Ag-L, created as a consequence of both RNA editing
and genome replication (19). In contrast, for Northern
analyses, we were able to detect genome replication as early as 2 days
(see Fig. 7).
Immunofluorescence.
Cell monolayers on glass coverslips were
fixed and stained as previously described (1). A human
anti-Ag antibody, conjugated with fluorescein (a gift of John
Gerin), was used to detect total Ag. For the detection of Ag-L, a
rabbit antipeptide antibody rab LP3 (a gift of Stan Lemon
[29]) was used in a combination with goat anti-rabbit
immunoglobulin G polyclonal antibody conjugated with rhodamine (Sigma).
To stain the cellular DNA, 2 µg of 4',6-diamidino-2-phenylindole (DAPI [Sigma]) was added to secondary antibody solutions. After mounting, the samples were viewed with a Zeiss Axiophot microscope with
a ×40 or ×100 objective and specific filter blocks. Images were
acquired with a charge-coupled device camera (Photometrics, model CH
250) and processed with Photoshop and Canvas software.
Northern blot analysis.
For studies of the initiation of
genome replication in transfected cells, total cellular RNA was
extracted with Tri Reagent (Molecular Research Center), glyoxalated,
and analyzed by electrophoresis as previously described (1).
RNA was transferred electrophoretically to a nylon membrane (Zeta-probe
GT; Bio-Rad) and immobilized by UV cross-linking. Hybridization was
performed with 32P-labeled RNA probes specific for genomic
HDV RNA, as described previously (1). Probe was synthesized
in vitro in the presence of [
-32P]UTP (DuPont).
Detection and quantitation of radioactivity were performed with a
phosphoimager (Fuji Bio-Imaging system).
Immunoblot analysis.
Total cell protein was analyzed under
denaturing conditions as described by Laemmli (15). After
electrotransfer to nitrocellulose, Ag was detected with a rabbit
polyclonal antibody followed by enhanced chemiluminescence (ECL;
Amersham).
Nucleotide sequence accession number.
The sequence of
plasmid pR5V5 has been assigned GenBank accession no. U88619.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Synthesis of Ag-S in E. coli.
Initial studies with
E. coli demonstrated poor expression of Ag-S from the
wild-type sequence. We noted that about 18% of the codons in the
natural Ag sequence are rarely used by E. coli. Others
have shown that attempted overexpression of codons that are rare in
E. coli not only can inhibit expression but also can lead to
misincorporation (7). Therefore, we designed a nucleotide sequence which maintained the amino acid sequence, but increased the
percentage of codons that were most favored for expression in E. coli from 26% to 85%. This optimized sequence (Fig. 1) was used
to construct expression plasmid pR5V5. We thus obtained a 40-fold
increase in expression and went on to purify the recombinant protein to
>85% homogeneity.
Recombinant Ag-S forms RNP with HDV RNA.
Experiments were
performed to determine whether the purified Ag-S could interact in
vitro with HDV RNA. Aliquots of 32P-labeled linear genomic
HDV RNA were incubated with increasing amounts of Ag-S. RNP assembly
was assayed by electrophoresis into agarose gels under nondenaturing
conditions, after which the gels were dried and the radioactivity was
quantitated with a phosphoimager.
Ag-S of 
100 nM (22 ng in 10 µl), the migration of the RNA was not significantly reduced relative
to that of free RNA (Fig. 3A). At 300 nM,
the RNA migration was reduced (Fig. 3B). Also, as the Ag-S
concentration was increased, the migration of the complexes was reduced
even further, and ultimately the complexes were barely able to enter
the gel (Fig. 3C to E).
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Transfection of cells with RNP assembled in vitro leads to genome
replication.
Each of the RNPs shown in Fig. 3 was tested in a
transfection assay. After 8 days, the total RNA was extracted and
subjected to Northern analysis to detect antigenomic (Fig.
4A) and genomic (Fig. 4B) RNA. At levels
of Ag-S in which no RNP formation had been previously observed (Fig.
3A), no replication was detected (Fig. 4, lanes 1 to 4). However, when
the level of Ag-S was sufficient for RNP formation (Fig. 3B to E),
there was significant accumulation of Ag and unit-length HDV RNAs in
the transfected cells (Fig. 4, lanes 5 to 9). No replication was
detected when genomic RNA was transfected alone (Fig. 4, lanes 10).
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Ag-S. This
was detected in the nucleus (Fig. 5A),
and the pattern was characteristic of HDV replication; that is, there was a diffuse nucleoplasmic staining as well as an accumulation of
small granules and larger discrete speckles, as observed previously (2). No signal was detected in cells transfected with either genomic RNA or Ag-S alone (data not shown).
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Ag-L. This assay represented an additional control,
because Ag-L was not present in the input RNP and could only be
produced by genome replication coupled with posttranscriptional RNA
editing (19). As shown in Fig. 5B, this antibody detected the characteristic nucleoplasmic signal, further confirming our interpretation that HDV genome replication was occurring.
In summary, these studies provided three lines of evidence that
recombinant Ag-S was necessary for the RNP, as assembled in vitro,
to be able to lead to genome replication when transfected into cells.
Are the events which lead to genome replication specific for
Ag-S?
It needs to be pointed out that the assay of transfection
with RNP as described above, involves not one but two
"sources" of functional Ag-S. The "early" source is
that which is present on the RNA as it is transfected into the
cell. The "late" source is de novo, in that it depends upon the
ensuing RNA-directed RNA replication, together with mRNA synthesis and
translation. Experiments were next carried out to determine whether the
initial source, the recombinant Ag-S, was specific, or whether other
RNA-binding proteins, including mutant forms of Ag-S, could
substitute.
Ag. These include multimerization ability,
high basicity, and a strong affinity for nucleic acids (6).
However, although the NC was able to form RNP with HDV RNA, as assayed
by gel electrophoresis (data not shown), transfection of cells with
these RNP complexes was unable to lead to detectable genome
replication, as assayed after 8 days, by immunofluorescence microscopy,
which is more sensitive than Northern analysis (data not shown).
Polylysine was also tested, because it is even more basic than Ag
and NC, and it also formed RNP complexes with HDV RNA, just as for
Ag-S in Fig. 3E (data not shown). Transfection with complexes made
with polylysine did not achieve detectable genome replication (data not
shown).
We next tested various truncated and modified forms of Ag (Fig. 2)
for their capacity to form RNP. Four were able to form RNP, as assayed
by gel electrophoresis, but on transfection failed to achieve genome
replication (data not shown): (i) a peptide consisting of the Ag
sequence aa 12 to 60 plus a C-terminal tyrosine (12-60Y) (17, 24,
30); (ii) another truncated version of Ag-S, this time with aa
1 to 84His6; (iii) a protein, His6Ag-L; (iv)
and a protein closely related to that of (iii) with the cysteine at
position 211 replaced by alanine. (In contrast to the wild-type Ag-L, this species could not be farneslyated, a modification which
is considered to enable packaging [22].)
A modified form of Ag-S whose transfection led to genome replication
was one with a C-terminal histidine tag. As detected by
immunofluorescence microscopy, the resultant replication level in
transfected cells (Fig. 6A) was
comparable to that obtained with wild-type Ag-S (Fig. 6B).
Comparable results were obtained with protein purified from Huh7 cells
rather than from E. coli; also functional was a Ag-S
modified with an N-terminal tag (data not shown).
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Ag-S, modified with either a C- or
N-terminal tag, in contrast to more extensively mutated forms, was able
to supply what we have defined as the early source of Ag-S. The data
support the interpretation that RNP formed by Ag-S and HDV RNA has
unique properties that, following transfection, lead to genome
replication.
Is de novo synthesis of Ag-S necessary for genome replication
initiated by transfection with RNP?
The studies mentioned above
showed that the early source of Ag-S, as delivered by transfection,
was necessary for achieving genome replication. We next asked whether
the same source might be sufficient for at least a limited amount of
genome replication. To remove the late or de novo source of Ag-S, we
replaced the linear multimeric HDV genomic RNA with a form that was
identical, except for a modification to block the open reading frame of
Ag-S.
Ag-S was still present at 2 days after transfection (data not shown). Therefore, cultures were transfected with either the
wild-type or mutated form of the HDV RNA, with or without prior
complexing with Ag-S. At 2 days the cell RNA was examined by
Northern analysis to detect antigenomic HDV RNA. As shown in Fig.
7 (lane 2), we only detected unit-length
antigenomic RNA in cells transfected with the wild-type RNA, as
delivered in a complex with Ag-S.
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Ag-S is necessary for
readily detectable levels of genome replication. However, they do not
exclude that with assays more sensitive than Northern analysis, some
levels of RNA-directed RNA synthesis might be detected.
Can the modified form of Ag-S replace the wild type as a late
source of Ag-S?
The studies described above showed that Ag-S
with a C-terminal histidine tag could replace the wild-type protein as
the early source of Ag-S. We next asked whether the same protein
could also provide the late de novo source. To answer this, we could not use a genome that encoded such a protein, because the associated genomic alterations would most likely have additional inhibitory consequences. Therefore, we made use of a cDNA cotransfection assay.
Cultures were transfected with a vector that expressed multimers of a
genome that was modified to block the open reading frame for Ag-S.
Huh7 cells were cotransfected with additional vectors, expressing
either the modified Ag-S protein, wild-type Ag-S as a positive
control, or Ag-L as a negative control. By immunoblotting, we found
that all three proteins were expressed (Fig.
8A). By Northern analysis, we found that
only the positive control supported genome replication; modified
Ag-S did not (Fig. 8B).
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that the modified Ag-S is sufficient early but not
sufficient lateraises the question of whether there is a difference
in the roles of Ag-S between the early and late effects. One model
is that there is a major difference; e.g., the early role may be simply
binding HDV RNA and transporting it to the nucleus. However, an
opposing model is that there is no qualitative difference; e.g., genome
replication involves the compound effect of many cycles, each with many
roles for Ag-S, so that even a minor deficiency in one or more roles
is ultimately compounded into a major effect on genome replication.
Further experiments are needed to distinguish between these models.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In a natural HDV infection, we expect that the virus binds to a
susceptible cell via an as yet unidentified receptor, is internalized, uncoated, and transported to the nucleus, and then RNA-directed RNA
synthesis is somehow initiated. These events take place as infection is
established in a susceptible animal (23) and could be
studied, not without difficulty, with cultures of primary human or
woodchuck hepatocytes (26, 27). We hope that our more
convenient receptor-independent transfection assay as described here,
proves to be a useful model for studying what we have defined as the "initiation of genome replication". This assay has some unique advantages relative to initiation of genome replication by cDNA transfection (14). It may even have a major advantage over
natural infections, in that we can readily manipulate in vitro, the
nature and composition of the RNP prior to the assay in vivo, of genome replication. In the present study, we have begun to test some variations for the RNP. For example, linear multimers of genomic RNA
could be replaced with antigenomic RNA (data not shown). Also successful was a range of stoichiometric amounts of Ag-S per RNA
(Fig. 4). The Ag-S was not only necessary but also specific, in that
several deleted forms of Ag-S or other RNA-binding proteins, were
not acceptable. In future studies, it will be possible to test other
variables, such as posttranslational modifications of the Ag-S,
different protein combinations, or modifications of the RNA, such as by
binding of oligonucleotides to specific regions that might be important
for the initiation of RNA-directed RNA synthesis.
In our transfection assay, if genome replication is ultimately detected
(either at 8 days by immunofluorescence microscopy [Fig. 5] and
Northern analysis [Fig. 4], or even at 2 days by Northern analysis
[Fig. 7]), then we can conclude that earlier, the transfected RNP was
used for RNA-directed RNA synthesis, leading to RNA processing and mRNA
synthesis. We say this because the genome replication, as defined,
depended not only an early source of Ag-S, as directly provided by
the transfection, but also on a de novo or late source of Ag-S (Fig.
7). Further experiments are needed to clarify the manner by which the
early source of Ag-S facilitates the initiation of genome
replication. Also, we need to know the extent to which these early
transcription and processing events taking place after RNP transfection
are a valid model of what happens in a natural infection. Currently, we
are examining the early RNA transcripts with procedures such as 5'
rapid amplification of cDNA ends, which are both more sensitive and
specific than Northern analyses, and we have begun to address such
important questions as defining the early role of Ag-S and determining the importance of the conformation (linear versus circular)
and local structure of the RNA template.
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ACKNOWLEDGMENTS |
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J.T. was supported by grants AI-26522 and CA-06927 from the NIH and by an appropriation from the Commonwealth of Pennsylvania. J.H. was supported by grant AI-32480 from the NIH.
Constructive comments on the manuscript were given by William Mason,
Glenn Rall, and Ting-Ting Wu. Antibodies essential to this work were
provided by John Gerin and Stan Lemon. The synthetic peptide 12-60Y was
also obtained from Stan Lemon. Patrick Brown provided the expression
plasmid for HIV-1 nucleocapsid protein. Vadim Bichko constructed
pVB101, -102, and -103; Matt Bockol constructed pMB001; and Ting-Ting
Wu constructed pTW203. The proteins HIV-1NCHis6 and
Ag-SHis6 were purified by Gloria Moraleda. Daphne Bell
and Binaifer Balsara assisted in the collection of charge-coupled device images.
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FOOTNOTES |
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* Corresponding author. Mailing address: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111-2497. Phone: (215) 728-2436. Fax: (215) 728-3616. E-mail: jm_taylor{at}fccc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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J Virol, June 1998, p. 4783-4788, Vol. 72, No. 6
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