Virology Group, International Centre for
Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi
110067, India
Hepatitis E virus (HEV) is a major human pathogen in the developing
world. In the absence of an in vitro culture system, very little
information on the basic biology of the virus exists. A small protein
(~13.5 kDa) of unknown function, pORF3, is encoded by the third open
reading frame of HEV. The N-terminal region of pORF3 is associated with
the cytoskeleton using one of its hydrophobic domains. The C-terminal
half of pORF3 is rich in proline residues and contains a putative
src homology 3 (SH3) binding domain and a mitogen-activated
protein kinase phosphorylation site. In this study, we demonstrate that
pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We
have isolated a 43-amino-acid interaction domain of pORF3 which is
capable of self-association in vivo and in vitro. The overlap of the
dimerization domain with the SH3 binding and phosphorylation domains
suggests that pORF3 may have a dimerization-dependent regulatory role
to play in the signal transduction pathway.
 |
TEXT |
Hepatitis E virus (HEV), the
causative agent of hepatitis E, is a waterborne pathogen which is
responsible for sporadic infections as well as large epidemics of acute
viral hepatitis in developing countries (4, 5, 16, 17, 18, 25,
26, 35). The HEV genome organization resembles that of many
alphaviruses, with nonstructural genes at the 5' end and structural
genes at the 3' end (18, 27). It has a single-stranded
positive-sense RNA genome of about 7.5 kb with three forward open
reading frames (ORF1, ORF2, and ORF3) encoding three different proteins
(19, 28, 31, 32). ORF1 (5,079 bp) is at the 5' end of the
genome and is predicted to code for putative nonstructural proteins
with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1, 15,
19). In the absence of a reliable culture system for HEV, fundamental studies on its replication and expression strategy have not
been undertaken. ORF2 and ORF3 have been expressed in Escherichia
coli, animal cells, baculovirus and yeast and in vitro in a
coupled transcription-translation system (10, 12, 20, 24).
ORF2 encodes the major HEV structural protein (pORF2), which has been
shown to be an 88-kDa glycoprotein that is expressed intracellularly as
well as on the cell surface. It is synthesized as a precursor and is
processed through signal sequence cleavage into the mature protein.
When expressed through the baculoviral expression system, pORF2 was
shown to assemble into virus-like particles which were cell associated
as well as secreted in the culture medium (16, 22, 33).
ORF3 encodes a small, 13.5-kDa cytoskeleton-associated phosphoprotein
(pORF3) (2, 36), which is expressed intracellularly and
shows no major processing. The location of ORF3 on the 3' end of the
genome classifies it as a structural protein, though its function is
yet to be defined.
In earlier expression studies, a dimeric form of pORF3 was always
observed by using polyacrylamide gel electrophoresis (12). In its primary sequence, pORF3 contains two distinct hydrophobic domains in its N-terminal region, amino acids 16 to 32 and 37 to 62. These may support homodimerization of the protein. We have used the
yeast two-hybrid system (7, 8) to show the
homodimerization of pORF3. We have subsequently identified the domains
of pORF3 that are involved in these homotypic interactions.
Surprisingly, the homodimerization domain maps in a single stretch of
43 amino acids at the C-terminal end of pORF3. This region contains a
smaller hydrophobic domain (amino acids 84 through 95) and two domains rich in proline residues. By constructing GAL4 activation and binding
domain fusions of truncated ORF3 proteins, we have shown, using the in
vivo yeast two-hybrid assay and in vitro immunoprecipitations, that
this 43-amino-acid interaction domain of pORF3 is capable of
self-association. The possible role of the homodimerization domain with
respect to its position on the ORF3 protein has been postulated.
In the few years since its introduction, the yeast two-hybrid system
has proven invaluable for studying physical interactions between
genetically defined partners, for identifying contacts among the
subunits of multiprotein complexes (6, 7, 11), and for
mapping specific domains involved in protein-protein interactions (14, 21, 30). In this system, two plasmid-borne gene
fusions are cotransformed into yeast cells and the interaction between two proteins is measured by the reconstitution of a functional transcriptional activator that triggers the expression of reporter genes lacZ and HIS3. We have used this system to
study the self-association of pORF3 by cloning the DNA binding domain
and the transcriptional activation domain upstream and in frame with
the ORF3 sequence.
Homotypic interactions of pORF3.
The full-length ORF3 of HEV
was excised from the pSG-ORF3 vector (12) by a
SmaI and BamHI digestion and cloned into the yeast two-hybrid vectors (Table 1),
resulting in an N-terminal in-frame fusion of either the GAL4 DNA
binding domain or the GAL4 activation domain to ORF3. All constructs
were verified by restriction digestion and sequencing. DNA
manipulations were carried out as described by Sambrook et al.
(29). Saccharomyces cerevisiae Y190
(MATa trp1-901 his3 leu2-3,112 ura3-52 ade2 gal4
gal80URA3::GAL-lacZ LYS2::GAL-HIS3) cells were transformed with
single plasmids or were cotransformed with the GAL4 BD and AD vectors
containing ORF3. The Y190 host strain containing pAS2-SNF1 and
pACT2-SNF4 was used as a positive control (9) and was
kindly supplied by Stephen Elledge. Y190 contains integrated copies of
both HIS3 and lacZ reporter genes under the
control of GAL4 binding sites. The results of the two-hybrid assay are
shown in Fig. 1. Single transformants
used in this assay were yeast (Y190) cells containing BD-ORF3, Y190
cells containing AD-ORF3, Y190 cells containing only the BD vectors,
and Y190 cells containing only the AD vectors (Fig. 1A). All these
transformants grew on the yeast extract-peptone-dextrose (YPD) plate
(nonselective media) (Fig. 1B). Single transformants containing the BD
by itself or as a fusion showed growth on the synthetic dextrose
Trp
(SD Trp) plate (Fig. 1C). Single
transformants containing the AD vector by itself or as a fusion showed
growth on the SD Leu plate (Fig. 1D). The cotransformants
were similarly plated on YPD and synthetic dextrose medium lacking Trp
or Leu or lacking Trp, Leu, and His (SD
TrpLeuHis), to select for
clones in which the HIS3 gene was transactivated. Growth of
the cotransformants in both SD Trp and SD
Leu plates simply showed that both plasmids were present
in the transformed cells. Growth of these clones on the SD
TrpLeuHis media showed
that the transcription of the HIS3 gene was turned on by the
reconstitution of the GAL4 transactivator due to a specific protein-protein interaction (Fig. 1E). Colonies were transferred to
nitrocellulose filters, and a 
-galactosidase filter assay was
performed as described earlier (3, 7) (Fig. 1F). The cotransformants containing both the BD-ORF3 and AD-ORF3 constructs along with the positive control used in this assay showed His prototrophy and -galactosidase activity on the filter assay.
View larger version (78K):
[in this window]
[in a new window]
|
FIG. 1.
Two-hybrid results showing full-length homotypic
interactions of the HEV ORF3 protein. (A) Template for panels B through
F, showing the clones streaked in each section of the plate. (B to E)
Growth on YPD, SD Trp, SD Leu, and SD
TrpLeuHis medium plates,
respectively. (F) Results from the -galactosidase filter assay.
Growth is represented by white on the black background of the petri
dish. Blue is represented by the black streaks on a white background
from nitrocellulose membrane.
|
|
The liquid -galactosidase assay was conducted and activity was
determined using the substrate chlorophenol
red--D-galactopyranoside as described previously
(3, 23). The host strain Y190 alone, along with single
transformants containing AD-ORF3 and BD-ORF3 and cotransformants
containing AD and BD vectors without a fusion protein, BD vector and
AD-ORF3, and BD-ORF3 and AD vectors, was used as a negative control
showing almost no -galactosidase activity. BD-SNF1/AD-SNF4 was the
positive control, and the clones containing BD-ORF3 and AD-ORF3 were
the test samples (Fig. 2). Relative
enzymatic activity was determined in five independent transformants
from each group. Our results from this assay indicate a strong
protein-protein interaction between the AD-ORF3 and BD-ORF3 proteins.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2.
Liquid -galactosidase assay results. Single
transformants and cotransformants were analyzed for a liquid
-galactosidase assay and were compared to each other. Values are
given in arbitrary units. The numbers above each bar represent the mean
of five independent transformants.
|
|
The specificity of the pORF3-pORF3 interaction was confirmed using a
yeast genetic assay for reconfirming positive two-hybrid interactions
(34). Plasmid constructs were extracted from the positive
Y190 cotransformants (BD-ORF3 and AD-ORF3, clone no. 1 and clone no. 2, shown in Fig. 1). The plasmids isolated from these clones were
separated and verified using E. coli HB101 cells on M9
synthetic media lacking Leu. Subsequently, these plasmids were singly
transformed into the PJ69-4a and PJ69-4
haploid yeast strains,
kindly provided by Philip James (13). After genetic crossing, the His3 protrophy of the diploid strains was tested by
plating for growth on SD His media. Only the diploids
containing both BD-ORF3 and AD-ORF3 showed a positive phenotype similar
to that for the positive control clone BD-SNF1/AD-SNF4 diploid (Fig.
3). All possible control transformations were conducted and were verified to be negative for His3 prototrophy, as shown in Fig. 3.
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 3.
Genetic verification of ORF3 homodimerization. The
haploid host cell is designated per its mating type, a or
. Diploid cells are designated a/. Growth of colonies is shown on
YPD (nonselective) and SD
TrpLeuHis media.
|
|
From the above experiments, it is clear that pORF3 forms a homodimer in
vivo. As mentioned earlier, there are distinct hydrophobic domains
present in ORF3 that may be responsible for homodimerization. Studies
on the molecular dissection of pORF3 were thus continued to map the
dimerization domain.
A 43-amino-acid region of the ORF3 protein is involved in
dimerization.
To characterize the homodimerization domain of the
ORF3 protein, various deletions were designed.
pGBT9-ORF3
56-123, pGAD424-ORF380-123, pGAD424-ORF31-80,
pGAD424-ORF31-80,100-123, and pGAD424-ORF31-100 were constructed
by restriction digestion and religation of the full-length constructs
GBT-ORF3 and GAD-ORF3 as described in Table 1.
Our two-hybrid experiments consisted of cotransformation of one of the
pORF3 truncated fusion proteins together with the corresponding fusion
protein containing either full-length or truncated ORF3. The results of
these experiments are shown in Fig. 4.
BD-ORF356-123 and AD-ORF380-123, when cotransformed with their
corresponding full-length ORF3 constructs (AD-ORF3 and BD-ORF3,
respectively), showed no reporter gene activity. However, the
ORF31-80 deletion, when cotransformed with its corresponding
full-length ORF construct (BD-ORF3), showed strong reporter gene
activation.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4.
Results of interactions of ORF3 deletion mutants with
full-length ORF3, in the yeast two-hybrid system. Shaded boxes
represent regions included in the respective constructs, whereas the
open boxes represent regions that were deleted from the wild-type
sequence. + signifies growth or blue color;  signifies no growth or
blue color. His represents growth on SD
TrpLeuHis media. The numbers
in brackets show relative -galactosidase units from the liquid
-galactosidase assay. AT signifies growth on SD
TrpLeuHis media with 50 mM
AT. Dip His represents growth of diploids tested through the genetic
two-hybrid assay.
|
|
We further investigated the strength of interactions by measuring the
HIS3 reporter gene for the full-length and truncated ORF3
proteins in the presence of 50 mM 3-aminotriazole (AT). Hundredfold serial dilutions of log-phase cultures of clones expressing BD-ORF3 and
AD-ORF3 and deletions of AD-ORF3 and BD-ORF3 and appropriate negative
controls were plated on SD His with 50 mM AT. These
results indicated the strength of the protein-protein interactions as a
function of His prototrophy. Cells containing both fusions of
full-length ORF3 proteins showed growth up to 104 serial
dilutions on the SD His 50 mM AT plate, confirming strong
homodimerization. The deletion ORF31-80 also showed growth at a
104 dilution, whereas BD-ORF356-123, AD-ORF380-123,
AD-ORF31-80,100-123, and AD-ORF31-100 showed no growth on SD
His 50 mM AT media. Results of this assay are shown in
Fig. 4, where + denotes growth of the clone being tested after a
104 dilution. This clearly showed that the interactions
between full-length pORF3 molecules were strong and that AD-ORF31-80
(containing amino acids 81 through 123) was sufficient to show a
protein-protein interaction (with its corresponding fusion full-length
BD-ORF3) of reporter strength equal to that of full-length ORF3 molecules.
Constructs pGAD424-ORF31-80,100-123 and pGAD424-ORF31-100 (Table
1) divide the 43-amino-acid C-terminal region of ORF31-80 into two
almost equal parts of about 20 amino acids each. When these two
constructs were tested against their corresponding BD-ORF3 full-length
fusion proteins for homodimerization, these two peptides were unable to
show a positive phenotype on the two-hybrid screen (Fig. 4). This
suggests that the 43-amino-acid region represents the intact
homodimerization domain. This 43-amino-acid binding domain contains a
hydrophobic domain (between amino acids 84 and 95) and also contains
two polyproline regions (amino acids 75 to 86 and 104 to 113). The
truncated proteins ORF31-100 and ORF31-80,100-123 divide the
43-amino-acid binding domain into two equal halves, each containing one
of the polyproline regions while the hydrophobic domain stays intact in
ORF31-80,100-123. This explains the liquid -galactosidase values
for a small stretch of 18 amino acids (ORF3 amino acids 81 to 99) fused
to AD, which were significantly higher (0.40) than those for its
counterpart AD-ORF31-100. Thus, we propose that the hydrophobic
domain within amino acids 84 through 95 is involved in dimerization but
is not enough to show a positive signal in the two-hybrid system.
It may be possible that the yeast two-hybrid system is unable to detect
an interaction as weak as this or that the fusion of approximately 140 amino acids (of GAL4) to an 18-amino-acid ORF3 region becomes
undetectable by the system, thus falling below the minimum threshold
required to turn on the HIS3 reporter significantly enough
to detect growth on selectable media. However, the 43-amino-acid region
(ORF amino acids 81 to 123) unequivocally shows homodimerization activity with full-length pORF3.
Self-association of the homodimerization domain.
Based on the
above observations, we constructed vectors expressing only the
43-amino-acid putative homodimerization domain as GAL4 AD and BD fusion
proteins. These constructs, BD-ORF31-80 and AD-ORF31-80, are
described in Table 1. Along with appropriate negative and positive
controls, we tested the dimerization activity of these constructs and
compared it to that for the full-length BD-ORF3 and AD-ORF3 positive
interaction clones. Cotransformants were obtained in yeast Y190 strains
containing the 43-amino-acid fusion constructs BD-ORF31-80 and
AD-ORF31-80. Cotransformants and the full-length ORF3
cotransformants (described previously) were simultaneously tested for
growth on YPD, SD Trp, SD Leu and SD
TrpLeu in order to confirm actively growing
cells and the presence of the BD vector, the AD vector, and both BD and
AD vectors, respectively.
Figure 5A through D shows the results of
our two-hybrid comparisons of full-length ORF3 homodimerization versus
a truncated ORF3 protein containing only the C-terminal 43-amino-acid
(81 to 123) homodimerization domain. Figure 5A and B show that both full-length and deletion clones are able to turn on the yeast two-hybrid reporter genes efficiently. Figure 5A shows the growth of
both full-length and truncated cotransformants on SD His
media. Figure 5B shows positive -galactosidase activity on the filter and liquid assays for both the full-length and 43-amino-acid fusion constructs. The quantitative liquid -galactosidase assay (results of which are shown in brackets) showed that the C-terminal 43-amino-acid region of ORF3 homodimerizes with an affinity almost equal to that of its wild-type full-length ORF3 counterpart. Figure 5C
shows the strength of the interactions measured on SD His
50 mM AT selective media, thus exhibiting the strength of the protein-protein interaction. Figure 5D indicates the ability of diploids to grow on selective media after a genetic cross of the singly
transformed haploid strains. This assay was conducted as described
previously (34).
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 5.
The 43-amino-acid domain of HEV ORF3 is capable of
self-association in vivo and in vitro. (A to D) In vivo results using
the yeast two-hybrid assay. Text in boldface describes the growth media
and/or assay used. The numbers in brackets in panel B represent
relative -galactosidase units in a liquid assay. (E) In vitro
results using a coupled transcription and translation assay system.
|
|
We also tested the homodimerization potential of full-length
pORF3 and the C-terminal 43 amino acids by means of an in vitro-coupled transcription-translation assay. The ORF3 full-length construct (pSGORF3, encoding 123 amino acids of pORF3) and the activation domain fusion ORF31-80 (pAD424-ORF31-80, encoding 156 amino acids
of the fusion protein) were used in a coupled in vitro
transcription-translation system (TNT coupled reticulocyte lysate
system; Promega) as per the manufacturer's instructions. When analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both
full-length ORF3 and the 43-amino-acid C-terminal ORF3 fusion proteins
showed homodimerization under fully denaturing conditions (with
-mercaptoethanol) and partially denaturing conditions (without
-mercaptoethanol) as shown in Fig. 5E. We have thus proved beyond
doubt that the interaction domain for homodimerization of HEV ORF3 lies
within amino acids 81 to 123.
The minor protein pORF3, encoded by ORF3 within the structural region
of the viral genome, is a 123-amino-acid protein of unknown function
(31). pORF3 has been shown to localize to the cytoplasm
and does not get posttranslationally modified (20, 36).
There are two large hydrophobic domains in the N terminus of ORF3, and
one of them has been shown to be responsible for association of pORF3
with the cytoskeleton. The hydrophobic domain which we find to be
involved in homodimerization is a clearly distinct region away from
these large hydrophobic domains and maps in the C-terminal region of ORF3.
pORF3 is also a phosphoprotein with a consensus phosphorylation
sequence between amino acids 78 and 83. Also, two polyproline regions
are present in the C-terminal region (amino acids 75 to 86 and 104 to
113) of pORF3. One of these has recently been shown to bind a number of
cellular proteins containing src homology 3 (SH3) domains
(S. Jameel, unpublished results). The dimerization domain that we have
shown in this study is also present in this C-terminal region of pORF3
(amino acids 81 through 123). The SH3 binding domains, the
phosphorylation signal, and the dimerization domains overlapping with
each other suggest that self-association of pORF3 may have a regulatory
function. It is known that the SH3 domains are found in many cellular
proteins involved in signal transduction pathways. Together with other
interaction domains, these are used to make critical protein-protein
contacts essential for signal transduction. The phosphorylation
of pORF3 by mitogen-activated protein kinase also suggests a
possible role for this viral protein in signal transduction. Its
association with the cytoskeleton using the N-terminal end of pORF3, as
well as a possible binding of SH3-containing targets to its C-terminal
end, suggests that homodimerization may have a regulatory role in
signal transduction by controlling the ability of pORF3 to interact
with SH3 domain-containing proteins. The steric possible hindrance
resulting from pORF3 dimerization may preclude pORF3-SH3 interactions
and thus preclude the downstream functions of pORF3 in the signaling pathway.
We gratefully acknowledge the generous gifts by Stephen Elledge of
the yeast two-hybrid vectors and strains and by Philip James of the
PJ69-4a and -4 yeast strains.
| 1.
|
Ansari, I. H.,
S. K. Nanda,
H. Durgapal,
S. Agrawal,
S. K. Mohanty,
D. Gupta,
S. Jameel, and S. K. Panda.
2000.
Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1).
J. Med. Virol.
60:275-283[CrossRef][Medline].
|
| 2.
|
Aye, T. T.,
T. Uchida,
X.-Z. Ma,
F. Iida,
T. Shikata,
H. Zhuang, and K. M. Win.
1992.
Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986-1988) of China.
Nucleic Acids Res.
20:3512[Free Full Text].
|
| 3.
|
Bai, C., and S. J. Elledge.
1996.
Gene identification using the yeast two-hybrid system.
Methods Enzymol.
273:331-347[CrossRef][Medline].
|
| 4.
|
Bradley, D. W.
1990.
Enterically-transmitted non-A, non-B hepatitis.
Br. Med. Bull.
46:442-461[Abstract/Free Full Text].
|
| 5.
|
Bradley, D. W., and M. A. Purdy.
1994.
Molecular and serological characteristics of hepatitis E virus, p. 42-45.
In
K. Nishioka, H. Susuki, S. Mishiro, et al. (ed.), Viral hepatitis and liver disease. Springer-Verlag, Tokyo, Japan.
|
| 6.
|
Brown, N. G.,
M. C. Costanzo, and T. D. Fox.
1994.
Interactions among three proteins that specifically activate translation of the mitochondria COX3 mRNA in Saccharomyces cerevisiae.
Mol. Cell. Biol.
14:1045-1053[Abstract/Free Full Text].
|
| 7.
|
Chien, C. T.,
P. L. Bartel,
R. Sternglanz, and S. Fields.
1991.
The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest.
Proc. Natl. Acad. Sci. USA
88:9578-9582[Abstract/Free Full Text].
|
| 8.
|
Fields, S., and O. Song.
1989.
A novel genetic system to detect protein:protein interactions.
Nature
340:245-246[CrossRef][Medline].
|
| 9.
|
Harper, J. W.,
G. R. Adami,
N. Wei,
K. Keyomarsi, and S. J. Elledge.
1993.
The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.
Cell
75:805-816[CrossRef][Medline].
|
| 10.
|
He, J.,
A. W. Tam,
P. O. Yarbough,
G. R. Reyes, and M. Carl.
1993.
Expression and diagnostic utilty of hepatitis E virus putative structural proteins expressed in insect cells.
J. Clin. Microbiol.
31:2167-2173[Abstract/Free Full Text].
|
| 11.
|
Jackson, A. L.,
P. M. Pahl,
K. Harrison,
J. Rosamond, and R. A. Sclafani.
1993.
Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.
Mol. Cell. Biol.
13:2899-2908[Abstract/Free Full Text].
|
| 12.
|
Jameel, S.,
M. Zafrullah,
M. H. Ozdener, and S. K. Panda.
1996.
Expression in animal cells and characterization of the hepatitis E virus structural proteins.
J. Virol.
70:207-216[Abstract].
|
| 13.
|
James, P.,
J. Halladay, and E. A. Craig.
1996.
Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.
Genetics
144:1425-1436[Abstract].
|
| 14.
|
Kalpana, G. V., and S. P. Goff.
1993.
Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system.
Proc. Natl. Acad. Sci. USA
90:10593-10597[Abstract/Free Full Text].
|
| 15.
|
Khudyakov, Y. E.,
M. O. Favorov,
D. L. Jue,
T. K. Hine, and H. A. Fields.
1994.
Immunodominant antigenic regions in a structural protein of the hepatitis E virus.
Virology
198:390-393[CrossRef][Medline].
|
| 16.
|
Khudyakov, Y. E.,
N. S. Khudyakova,
H. A. Fields,
D. Jue,
C. Starling,
M. O. Favorov,
K. Krawczynski,
L. Polish,
E. Mast, and H. Margolis.
1993.
Epitope mapping in proteins of hepatitis E virus.
Virology
194:89-96[CrossRef][Medline].
|
| 17.
|
Khuroo, M. S.
1980.
Study of an epidemic of non-A, non-B hepatitis: possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B type.
Am. J. Med.
68:818-823[CrossRef][Medline].
|
| 18.
|
Khuroo, M. S.,
M. R. Teli,
S. Skidmore,
M. A. Sofi, and M. Khuroo.
1981.
Incidence and severity of viral hepatitis in pregnancy.
Am. J. Med.
70:252-255[CrossRef][Medline].
|
| 19.
|
Koonin, E. V.,
A. E. Gorbalenya,
M. A. Purdy,
M. N. Rozanov,
G. R. Reyes, and D. W. Bradley.
1992.
Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-stranded RNA plant and animal viruses.
Proc. Natl. Acad. Sci. USA
89:8259-8263[Abstract/Free Full Text].
|
| 20.
|
Lal, S. K.,
P. Tulasiram, and S. Jameel.
1997.
Expression and characterization of the hepatitis E virus ORF3 protein in the methylotrophic yeast, Pichia pastoris.
Gene
190:63-67[CrossRef][Medline].
|
| 21.
|
Lalo, D.,
C. Carles,
A. Sentenac, and P. Thuriaux.
1993.
Interactions between three common subunits of yeast RNA polymerases I and III.
Proc. Natl. Acad. Sci. USA
90:5524-5528[Abstract/Free Full Text].
|
| 22.
|
Li, T. C.,
Y. Yamakawa,
K. Suzuki,
M. Tatsumi,
M. A. Razak,
T. Uchida,
N. Takeda, and T. Miyamura.
1997.
Expression and self-assembly of empty virus-like particles of hepatitis E virus.
J. Virol.
71:7207-7213[Abstract].
|
| 23.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 24.
|
Panda, S. K.,
I. H. Ansari,
H. Durgapal,
S. Agrawal, and S. Jameel.
2000.
The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.
J. Virol.
74:2430-2437[Abstract/Free Full Text].
|
| 25.
|
Purcell, R. H., and S. U. Emerson.
2000.
Hepatitis E virus infection.
Lancet
355:578.
|
| 26.
|
Purcell, R. H., and J. R. Ticehurst.
1988.
Enterically transmitted non-A, non-B hepatitis: epidemiology and clinical characteristics, p. 131-137.
In
A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York, N.Y.
|
| 27.
|
Purdy, M. A.,
A. W. Tam,
C. C. Huang,
P. O. Yarbough, and G. R. Reyes.
1993.
Hepatitis E virus: a non-enveloped member of the `alpha-like' RNA virus supergroup?
Semin. Virol.
4:319-326[CrossRef].
|
| 28.
|
Reyes, G. R.,
C. C. Huang,
A. W. Tam, and M. A. Purdy.
1993.
Molecular organization and replication of hepatitis E virus (HEV).
Arch. Virol.
7:15-25.
|
| 29.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory mannual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 30.
|
Takacs, A. M.,
T. Das, and A. K. Banerjee.
1993.
Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system.
Proc. Natl. Acad. Sci. USA
90:10375-10379[Abstract/Free Full Text].
|
| 31.
|
Tam, A. W.,
M. M. Smith,
M. E. Guerra,
C. C. Huang,
D. W. Bradley,
K. E. Fry, and G. R. Reyes.
1991.
Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome.
Virology
185:120-131[CrossRef][Medline].
|
| 32.
|
Tsarev, S. A.,
S. U. Emerson,
G. R. Reyes,
T. S. Tsareva,
L. J. Letgers,
I. A. Malik,
M. Iqbal, and R. H. Purcell.
1992.
Characterization of a prototype strain of hepatitis E virus.
Proc. Natl. Acad. Sci. USA
89:559-563[Abstract/Free Full Text].
|
| 33.
|
Tuteja, R.,
T. C. Li,
N. Takeda, and S. Jameel.
2000.
Augmentation of immune responses to hepatitis E virus ORF2 DNA vaccination by codelivery of cytokine genes.
Viral Immunol.
13:169-78[Medline].
|
| 34.
|
Tyagi, S., and S. K. Lal.
2000.
Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system.
Biochem. Biophys. Res. Commun.
277:589-593[CrossRef][Medline].
|
| 35.
|
Wong, D. C.,
R. H. Purcell,
M. A. Sreenivasan,
S. R. Prasad, and K. M. Pavri.
1980.
Epidemic and endemic hepatitis in India: evidence for a non-A, non-B virus etiology.
Lancet
ii:876-879.
|
| 36.
|
Zafrullah, M.,
M. H. Ozdener,
S. K. Panda, and S. Jameel.
1997.
The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.
J. Virol.
71:9045-9053[Abstract].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Text
