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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). 304 3 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 [1] Amarasinghe, G.K., De Guzman, R.N., Turner, R.B., Chanecellor, K.J., Wu Z.R., and Summers, M.F., NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi -RNA packaging signal. implication for genome recognition, J. Mol. Biol., 301:491–511, 2000. [2] Urbaneja, M.A., McGrath, C.F., Kane, B.P., Henderson, L.E., and Casas-Finet, J.R., Nucleic acid binding properties of the simian immnodeficiency virus nucleocapsid protein NCp8, J. Biol. Chem., 275(14):10394–10404, 2000. [3] Zuker, M., The MFOLD Program in the Institute for Biochemical Computing Washington University School of Medicine, USA, version 3.0. http://mfold2.wustl.edu/ mfold/rna/form1.cgi [4] De Guzman, R.N., Wu, Z.R., Stalling, C.C., Pappalardo, L, Borer, P.N., Summers, M.F., Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element, Science, 279:384–388, 1998. [5] Rizvi, T.A. and Panganiban, A.T., Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles, J. Virol., 67(5):2681–2688, 1993. [6] Guan, Y., Whitney, J.B., Diallo, K., and Wainberg, M.A., Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus, J. Virol., 74(19):8854–8860, 2000.