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J. Biol. Chem., Vol. 277, Issue 25, 22759-22767, June 21, 2002
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From the Virology Group, International Centre for Genetic
Engineering and Biotechnology, Aruna Asaf Ali Road,
New Delhi 1100067, India
Hepatitis E virus (HEV) is a human RNA virus
containing three open reading frames. Of these, ORF1
encodes the viral nonstructural polyprotein; ORF2 encodes
the major capsid protein, which exists in a glycosylated and
non-glycosylated form; and ORF3 codes for a phosphoprotein
of undefined function. Using fluorescence-based colocalization, yeast
two-hybrid experiments, transiently transfected COS-1 cell
co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2
protein. The domains involved in this ORF2-ORF3 association have
been identified and mapped. Our deletion analysis showed that a
25-amino acid region (residues 57-81) of the ORF3 protein is
required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase
digestion assay, we showed that the ORF2-ORF3 interaction is dependent
upon the phosphorylation at Ser80 of ORF3. Finally, using
COS-1 cell immunoprecipitation experiments, we found that the
phosphorylated ORF3 protein preferentially interacts with the
non-glycosylated ORF2 protein. These findings were confirmed using
tunicamycin inhibition, point mutants, and deletion mutants expressing
only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational
modification-dependent manner. Such an interaction of ORF2
with ORF3 suggests a possible well regulated role for ORF3 in HEV
structural assembly.
The Phosphorylated Form of the ORF3 Protein of Hepatitis E Virus
Interacts with Its Non-glycosylated Form of the Major Capsid Protein,
ORF2*
,
*
This work was supported by the International Centre for
Genetic Engineering and Biotechnology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Research Fellow from the University Grants Commission.
§
To whom correspondence should be addressed. Tel.: 91-11-6177357;
Fax: 91-11-6162316; E-mail: sunillal@icgeb.res.in.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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