Download RNA Secondary Structure Based Prediction of Simian

Genome Informatics 11: 303–304 (2000)
303
RNA Secondary Structure Based Prediction of Simian
Immunodeficiency Virus Nucleocapsid NCp8 Protein
Recognition Site
Naoko Takada
Yukio Sugiura
[email protected]
[email protected]
Institute for Chemical Research, Kyoto University, Gokasyo, Uji, Kyoto 611-0011,
Japan
Keywords: nucleocapsid protein, simian immunodeficiency virus (SIV), RNA binding specificity
1
Introduction
Efficient and specific encapsidation of retroviral RNA by the assembling virion particle is an essential
step in the retrovirus life cycle. In this process, full length genomic RNA is preferentially packaged,
whereas spliced viral RNA and cellular RNA are generally excluded from nascent particles. There
are two elements necessary for specific packaging of viral RNA. One of them, cis-acting sequence, is
recognition site located in the 5’- and/or 3’- untranslated genomic RNA, and the other, trans-acting
element, is nucleocapsid protein NCp of Gag protein. NCps have highly conserved “zinc finger motif”,
Cys-x2-Cys-x4-His-x4-Cys. The zinc finger motif contributes to sequence-specific binding to viral
RNA.
Human immunodeficiency virus type 1 (HIV-1) NCp7 recognizes the psi region of 5’-leader HIV-1
genome. The psi region has four stem-loops, SL1, SL2, SL3, and SL4. NCp7 binds both SL2 and SL3
with high affinity [1]. The NMR structure of NCp7 and SL3 complex is available (Protein Data Bank
accession number is 1A1T) [4]. In other retroviruses, NCps would also bind stem-loop structures of
genomic RNA.
NCp8 of the simian immunodeficiency virus (SIV) is highly homologous with NCp7 in amino acid
sequence. Several subtypes of SIV genome have been sequenced, and NCp8 also binds to package
5’-leader RNA during viral assembly but limited binding regions are not known. NCp8 is assumed to
bind stem-loops in 5’-leader SIV genomic RNA. Here, we estimated the RNA region that can make
stable stem-loop structures by means of RNA secondary structure prediction, and investigated the
binding affinities of NCp8 to the candidate stem-loops.
2
Computational Method
The genomic sequences of SIV subtypes were derived from Genbank database, version 119.0. Multiple
alignments of the 5’-leader sequences were performed with CLUSTALW to get specified conserved
regions. The predicted secondary structures of 5’-leader sequence of SIV RNA were obtained with the
MFOLD program by dynamic programming [3]. In addition, the secondary structure of the HIV-1 psi
region was predicted to confirm the reliability of MFOLD.
In order to obtain the energy-minimized complex of NCp8 and target RNA, we have done the
homology modeling by using 1A1T, the complex of HIV-1 NCp7 and SL3, as a template. These
homology modeling were realized by the energy minimization process with the Insight II software
package (MSI) utilizing a modified Consistent Valence Force Field (CVFF).
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Takada and Sugiura
Experimental Method
The nucleocapsid proteins, NCp7 and NCp8, are prepared by Fmoc (9-fluorenylmethoxycarbonyl)
strategy and purified by HPLC (High - Performance Liquid Chromatography). The amino acid sequence of NCp7 is derived from HIV-1pNL4-3 used in 1A1T. The NCp8’s amino acid sequence is
derived from SIV-subtype (SIV-Mne) used in the preceding research [2]. The primary sequences of
the peptides were verified by means of mass spectrometry. The candidate RNA fragments predicted
by MFOLD and HIV-1 SL3 were synthesized by in vitro transcription.
In order to determine the binding affinity between the NCp8 and the RNA stem-loop fragments, gel
shift experiments were performed. We used NCp7 and SL3 as a control. The NCps and the 5’-labelled
RNA stem-loops were incubated and the reaction mixtures were loaded to gel electrophoresis. The
radioactive signals were quantitated by autoradiography, and apparent dissociation constants were
determined.
4
Results and Discussions
The secondary structure prediction of the HIV-1 psi region revealed four stem-loops of SL1, SL2, SL3
and SL4. Then, we determined to application of MFOLD to SIV RNA. The multiple alignment of SIV
5’-leader sequence showed that there are highly conserved regions among SIV subtypes. From these
regions, the secondary structures were predicted to find several stem-loops. Gel shift assay showed
that some SIV candidate stem-loops had relatively high binding affinity with NCp8. Although further
experiments are required, they are considered to be one of the NCp8 recognition site.
In the case of DNA, the primary sequence is the only factor to define its function because of the
unique structure. The way to predict DNA function has been almost established on the basis of the
primary sequence. On the other hand, functional RNA has secondary and tertiary structures. Thus,
RNA primary sequence doesn’t reflect its function directly. In this study, we performed structure
based prediction by taking advantage of one of the simplest RNA secondary structures, stem-loop.
As a result, we found functional RNA binding to NCp8. These results presented the possibility of
applying computational methodology to investigation of broad field.
Acknowledgement
Computational time was provided by the Supercomputer Laboratory, Institute for Chemical Research,
Kyoto University, Japan.
References
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