Silvy 73 Profile of nonstructural glycoprotein NS1 as a diagnostic marker in dengue type 2 virus infection Sylvia Y. Muliawan* ABSTRACT BACKGROUND Several studies in dengue on cloned or cell culture passaged in wild and attenuated virus have been performed, but each report described different sites of nucleotide and amino acid changes possibly associated with virulence. The aim of this study was to investigate protein expression of dengue type 2 virus infected C6/36 cells through two-dimensional gel electrophoresis followed by N-terminal Edman degradation sequencing to identify target proteins. METHODS Two type of samples; dengue type 2 virus-infected C6/36 cells and uninfected C6/36 cells were prepared. Immobilized non-linear pH gradient strips, pH 3-10 were used in isoelectric focusing, and 10% homogeneous gels were used in Sodium dodecyl sulphate - Polyacylamide gel electrophoresis. The target protein spots were subsequently transferred onto polyvinyldifluoride membrane by western blotting and visualized by coomassie brilliant blue for N-terminal sequencing purpose. RESULTS This study revealed four target protein spots in dengue type 2 virus-infected C6/36 cells that were not present in uninfected C6/36 cells. The N-terminal sequencing result was D-S-G-C-V-V-S-W-K-N-K which was identical to nonstructural glycoprotein NS1 (Swissprot-database) associated with the replication process of flaviviruses. CONCLUSION Nonstructural NS1 might be used as a diagnostic marker and/or as a parameter to evaluate the effect of antiviral agents for dengue type 2 virus infection/replication. Keywords: Dengue type 2 virus, non structural NS1 protein, proteomics *Department of Microbiology Medical Faculty Trisakti University Correspondence Dr. Sylvia Y. Muliawan, PhD Department of Microbilogy Medical Faculty Trisakti University Jl. Kyai Tapa No.260, Grogol Jakarta 11440 Telp. 5672731 eks.2402 Email: sylviaym2003@yahoo.com Universa Medicina 2007; 26: 73-9. UNIVERSA MEDICINA April-Juni 2007April-Juni 2007April-Juni 2007April-Juni 2007April-Juni 2007 Vol.26 - No.2 Vol.26 - No.2 Vol.26 - No.2 Vol.26 - No.2 Vol.26 - No.2 74 Muliawan Nonstructural glycoprotein NS1 Profil glikoprotein “non structural” NS1 sebagai marker pada diagnosis infeksi virus dengue tipe 2 Sylvia Y. Muliawan LATAR BELAKANG Pada dengue telah dilakukan beberapa penelitian yang menggunakan cara cloning atau subkultur sel baik yang berasal dari virus yang delemahkan maupun yang tidak dilemahkan, tetapi masing-masing hasil memperlihatkan perubahan asam amino dan nukleotida pada tempat yang berbeda yang berhubungan dengan virulensi. Tujuan penelitian adalah untuk menyelidiki ekspresi protein virus dengue tipe 2 yang di kultur pada sel C6/36 melalui two-dimensional gel electrophoresis. CARA Sampel yang digunakan terdiri dari dua tipe, yaitu kultur sel C6/36 yang diinfeksi dengan virus dengue tipe 2 dan kultur sel C6/36 yang tidak diinfeksi. Pada teknik isoelectric focusing digunakan immobilized non-linear pH gradient strips dengan pH 3-10 yang diikuti dengan Sodium dodecylsulphate-Polycrylamide gel electrophoresis. Selanjutnya protein target yang diperoleh dipindahkan ke polyvinyldifluoride membrane dengan metode Western blot dan dilakukan pewarnaan dengan coomassie brilliant blue untuk keperluan N-terminal sequencing. HASIL Hasil yang diperoleh pada ekspresi protein adalah 4 titik ekstra yang ditemukan pada sel C6/36 yang diinfeksi virus dengue tipe 2, tetapi tidak ditemukan pada sel C6/36 yang tidak diinfeksi. Hasil N-terminal sequencing menunjukkan urutan sekuens yang identik dengan nonstructural glycoprotein sesuai dengan Swissprot-database yaitu D-S-G-C-V-V-S-W-K-N-K. Sekuens ini berkaitan dengan proses replikasi flaviviruses. KESIMPULAN Nonstructural NS1 dapat digunakan sebagai alat bantu diagnosis dan atau parameter untuk mengevaluasi efek antivirus terhadap infeksi atau proses replikasi virus dengue tipe 2. Kata kunci: Virus dengue tipe 2, protein non structural NS1, proteomik *Bagian Mikrobiologi Fakultas Kedokteran Universitas Trisakti Korespondensi Dr. Sylvia Y. Muliawan, PhD Bagian Mikrobiologi Fakultas Kedokteran Universitas Trisakti Jl. Kyai Tapa No.260, Grogol Jakarta 11440 Telp. 5672731 eks.2402 Email: rsylviaym2003@yahoo.com Universa Medicina 2007; 26: 73-9. ABSTRAK INTRODUCTION Dengue fever (DF), dengue hemorrhagic fever (DHF) and shock syndrome (DSS) are mosquito-borne infectious diseases that have b e c o m e m a j o r i n t e r n a t i o n a l p u b l i c h e a l t h concerns. DF and DHF/DSS occur in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas.(1) A c c o r d i n g t o Wo r l d H e a l t h O rg a n i z a t i o n (WHO) estimates, its incidence has increased by a factor of 30 over the last 50 years.(2) It occurs in tropical areas and affect up to 100 million people each year,(3,4) including 500,000 75 cases of DHF and around 30,000 deaths, mostly among children.(5) It is now endemic in more than 100 countries (the Americas, the eastern Mediterranean, Southeast Asia, and the Western Pacific) and poses a threat to more than 2.5 billion people.(6) The African continent seems less affected by the dengue virus.(5) An epidemic o f D H F i n t h e N e w Wo r l d t o o k p l a c e i n Venezuela in 1989; isolates of DEN-1, DEN-2 and DEN-4 were obtained during this epidemic, but the most severe cases were associated with DEN-2 infections.(10) Dengue viruses belong to the Flavivirus genus of the Flaviviridae family. Flaviviruses are enveloped, single stranded R N A v i r u s e s . T h e d e n g u e g e n o m e i s approximately 11 kb in size and contains a single open reading frame, which encodes a precursor polyprotein and is flanked by two nontranslated regions (5’ and 3’ NTR). Co- and posttranslational proteolytic cleavage of the precursor results in the formation of three structural proteins, capsid (C), membrane (M), and envelope (E), and seven nonstructural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, N S 4 B , a n d N S 5 . A m o n g t h e m , t h e nonstructural protein NS1 related to replication process of flaviviruses. Unusually for a viral glycoprotein, NS1 is produced both membrane- associated and secreted forms(5) both of which are highly immunogenic and can elicit high-titre antibody.(11) NS1 has a predicted molecular mass of 46 to 50 kilodalton(12) due to N-linked carbohydrate chains at position 130 and 207 and 12 invariant cysteine residues.(13) NS1 is not incorporated into the virion and therefore does not elicit virus-enhancing antibodies.(14) The precise function of dengue NS1 protein remains unclear.(15) P r o t e o m i c s i s w i d e l y a c c e p t e d a s a technology in the post genomic era to identify proteins that are either involved in a specific cellular process (cell map proteomics) or exhibit an altered expression profile as the r e s u l t o f s o m e c h a n g e s i n p h y s i o l o g i c a l c o n d i t i o n ( e x p r e s s i o n p r o t e o m i c s ) . S i n c e proteins play a central role in the life of an o r g a n i s m , p r o t e o m i c s i s i n s t r u m e n t a l i n discovery of biomarkers, such as markers that indicate a particular disease. T h e d e v e l o p m e n t o f t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s ( 2 - D E ) w a s a m a j o r s t e p forward of the science in proteomics to identify the proteins of interest. The 2-DE is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissue, or other biological samples. (16) One problem that had to be overcome was the lack of sensitive protein sequencing technology. The f i r s t m a j o r t e c h n o l o g y t o e m e rg e f o r t h e identification of proteins was the sequencing of proteins by Edman degradation.(17) Followed by image analysis and mass spectrometry to quantitify and to characterize proteins.(18) The aim of this study was to investigate protein expression of dengue type 2 virus-infected C6/ 3 6 c e l l s t h r o u g h t w o - d i m e n s i o n a l g e l electrophoresis followed by N-terminal Edman d e g r a d a t i o n s e q u e n c i n g t o i d e n t i f y t a rg e t proteins. MATERIAL AND METHODS Preparation of uninfected sample C 6 / 3 6 c e l l s f r o m t h e D e p a r t m e n t o f Medical Microbiology, Faculty of Medicine, U n i v e r s i t y o f M a l a y a , M a l a y s i a w e r e m a i n t a i n e d i n R P M I - 1 6 4 0 m e d i u m ( F l o w Laboratories, U.K.) supplemented with 2% fetal calf serum, and incubated at 28°C. The C6/36 cells were disrupted by repeated fast freezing, three times for 15 minutes, and subsequently pelleted by spinning at 4°C, 2000 rpm for 10 minutes. The pellet was twice washed with 1 ml cold-PBS (Phosphate saline buffer), spun as above, then resuspended with 1 ml lysis buffer (8M urea, 4% Triton-X 100, Universa Medicina Vol.26 No.2 76 Muliawan Nonstructural glycoprotein NS1 2% IPG-phor buffer 3-10), incubated for 1 hour at 4°C and again spun as above. The supernatant was stored at -20°C. DV2-infected C6/36 cells Dengue virus-2 (New-Guinea C strain) was obtained from the Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia. Virus stocks were used to infect 80% confluent cell monolayers in RPMI- 1640 supplemented with 2% fetal calf serum and incubated at 28°C until cytopathic effect (CPE) was observed (up to day-4), at which stage the supernatant and cell monolayers were harvested. Cell processing used the same method as described for uninfected cells. Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis (2- DGE) was performed using the Multiphore II E l e c t r o p h o r e s i s S y s t e m b a s e d o n t h e r e c o m m e n d e d m e t h o d b y A m e r s h a m Biosciences.(19) Sample was diluted in buffer containing 8M urea, 0.5% (v/v), Triton X-100, 0.5% (v/v), IPG-phor buffer 3-10 and 12 mM dithiothreitol (DTT). Pre-cast immobilized dry strips pH 3-10 (Amersham Biosciences) were rehydrated overnight in the same solution. The strips were aligned in a tray that was filled with mineral oil (Amersham Biosciences). Isoelectric focusing was performed in three steps by varying the voltage as suggested by the manufacturer. For the second dimension, the strips were incubated for 15 minutes in a solution consisting of 6M Urea, 1% (w/v) SDS, 30% (v/v) glycerol and 0.3M DTT dissolved in 0.05M Tris-HCl (pH 6.8). This was followed by incubation for another 15 minutes in a similar solution but containing 50mM iodoacetamide instead of DTT. St r i p s w e r e p l a c e d o n a S o d i u m d o d e c y l sulphate-Polyacylamide gel electrophoresis (SDS-PAGE) 10% homogeneous gel (Amersham Biosciences) and electrophoresed for 100 minutes. Visualization step was done by silver staining. For determination of molecular weight of the target proteins, marker with low range w a s r u n t o g e t h e r o n S D S - PA G E w i t h t h e molecular weight kit in range of 45 kDa and 66 kDa (Bio-Rad manufacturer). Immundblotting and immund detection Western blotting was performed according to the standard method.(20) Nova Blot Kit of M u l t i p h o r e I I E l e c t r o p h o r e s i s S y s t e m (Pharmacia, Uppasala, Sweden) was used to transfer the protein to nitrocellulose membrane (Protran-Schlicher and Schuell-0.45 µM). The unit was run at 0.8mA/cm2 for 2 hours. After transfer was completed, immunodetection procedure was performed using hyperimmune anti rabbit sera against DV2 and amplified a l k a l i n e p h o s p h a t a s e g o a t a n t i - r a b b i t immunoblot assay kit (Bio-Rad). The reaction was stopped by washing the membrane with distilled water. Preparation of sample for Edman sequencing Western blotting was performed as per standard method.(20) Nova Blot Kit of Multiphore I I E l e c t r o p h o r e s i s S y s t e m ( P h a r m a c i a , Uppasala, Sweden) was used to transfer the p r o t e i n t o p o l y v i n y l d i f l u o r i d e ( P V D F ) membrane (0.45 µm). To vasilitate the N- terminal sequencing process, the infected C6/36 cells were run together on SDS-PAGE with the molecular weight kit. After wards, the protein spots from SDS-PAGE were transferred to PVDF membrane and visualized with coomassie brilliant blue staining (Figure 2), then sent for N-terminal Edman degradation sequencing (Midwild Analytical, USA). RESULTS T h i s i n v e s t i g a t i o n o f d e n g u e p r o t e i n expression revealed four target protein spots on 77 DV2-infected C6/36 (Figure 1a), but not on uninfected C6/36 (Figure 1b). The result of immunoblotting and immunodetection of infected C6/36 cells showed the target protein spots (Figure 2a) were picked by antibody against DV- 2 and were visualized as a purple color (Figure 2b). The molecular weight of the protein spots in infected C6/36 cells was 50 kDa in (Figure 3a) and immunoblotting of infected C6/36 cells onto PVDF membrane with coomassie blue staining for N-terminal sequencing purpose was s h o w e d F i g u r e 3 b . N-terminal sequencing determined the protein spots to be D-S-G-C-V- V-S-W-K-N-K (Midwild-Analytical,USA), which was identical to nonstructural glycoprotein NS1 ( S w i s s p r o t - d a t a b a s e ) a s s o c i a t e d w i t h t h e replication process of flaviviruses. DISCUSSION In this study, the aim of this study was to observe the protein expression of uninfected and infected C6/36 cells by DV2 through 2-DGE. This study found protein spots in DV2-infected C6/36 cells which were absent in uninfected C6/ 36 cells (Figure 1). Immunoblotting was done to prove the target protein spots originally came from DV2 infection. Based on Swiss-prot database, the 50 kDa protein spots turned out to have the following sequence: D-S-G-C-V-V- S-W-K-N-K, which was determined to be homologous to non structural NS1. Glycoprotein NS1, present in all flaviviruses, appears to be essential for virus viability. NS1 is produced both membrane-associated an secreted forms(5) both of which are highly immunogenic and can elicit high-titre antibody.(11) This is presumably because NS1 is present on the surface of infected cells and is efficiently secreted.(21) In the post genomic era, proteins are coming back into focus because it has been realized again than whole genome sequence information alone is not s u ff i c i e n t t o e x p l a i n a n d p r e d i c t c e l l u l a r phenomena, as it is largely the proteins that execute and control the majority of cellular activities. There are several reasons for focusing on the analysis of proteins, such as the level of mRNA expression frequently does not represent the amount of active protein in a cell(22) or the g e n o m e s e q u e n c e d o e s n o t d e s c r i b e p o s t t r a n s l a t i o n a l m o d i f i c a t i o n , w h i c h m a y b e essential for protein function and activity. In this study, the protein spots did not migrate to the s a m e l e v e l , p r o b a b l y d u e t o t h e d i ff e r e n t g l y c o s y l a t i o n p a t t e r n s . ( 1 3 ) N S 1 p r o t e i n expression through proteomics technique might be of use as a diagnostic marker and/or as a parameter for evaluation of the efficacy of antiviral agents in DV2 infection. The value of using NS1 as a surrogate marker of infection is supported by Young et al who assessed the potential of capture ELISA as a diagnostic assay(23) and Huang et al who showed high level expression of recombinant dengue viral NS1 protein used as a diagnostic antigen.(24) There are no known reports from other researchers on this protein using identical or similar techniques. CONCLUSION The application of NS1 separated by 2- DGE may provide an alternative approach for monitoring or as a parameter for evaluation the e ff i c a c y o f a n t i - v i r a l a g e n t s a g a i n s t D V 2 infection/replication. ACKNOWLEDGEMENTS The author expresses her gratitude to Prof. Lam Sai Kit, Prof. Shamala Devi, Prof Onn Hashim, Prof. Rohana Yusof from Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia and also to Associate Prof. Dr. Thomas J. Chambers, Saint Louis University School of Medicine, St.Louis, USA for their keen interest and support in this study. Universa Medicina Vol.26 No.2 78 Muliawan Nonstructural glycoprotein NS1 Figure 1. Protein expression of (a) Infected C6/36 cells with DV2 and (b) Uninfected C6/36 cells (a) (b) Figure 2. Protein expression of (a) Infected C6/36 cells with DV2 and (b) Immunoblotting and immunodetection of infected C6/36 cells with specific antibody against DV2 onto nitrocellulose membrane (a) (b) 97kDa 66kDa 45kDa 30kDa Figure 3. Protein expression of (a) Infected C6/36 cells with the molecular weight of target protein spots and (b) immunoblotting of infected C6/36 cells onto PVDF membrane with coomassie blue staining (a) (b) 79 References 1. Cisneros A, Diaz-Bdillo A, Cruz-Martinez G, Tovar R, Amirez-Palacios LR, Jimenez-Rojaz F, et al. Dengue 2 genotypes in the state of Oaxaca, Mexico. Arch Virol 2006; 151:113-25. 2. World Health Organization. Epidemic and pandemic alert and response impact of dengue. World Health Organization, Geneva, Switzerland 2005. Available at: http:/www.who.int/csr/disease/ dengue/impact/en/index.html. Accessed 18 April, 2006. 3. Gubler D. Dengue virus and dengue hemorrhagic fever. J Clin Microbiol Rev 1998; 11: 480-96. 4. McBride WJH, Ohmann HB. Dengue viral infections; pathogenesis and epidemiology. Microbes Infect 2000; 2:1041-50. 5. Dussart P, Labeau B, Lagathu G, Louis P, Nunes MRT, Rodrigues SG, et al. Evaluation of an Enzyme Immunoassay for detection of dengue virus NS1 antigen in human serum. Clin and Vac Immunol 2006; pp.1185-9. 6. Guzman MG, Kourl G. Dengue: an update. Lancet Infect Dis 2002; 2:33-42. 7. Kalayanarooj S, Vaughn DW, Nimmannitya S, Green S, Suntayakorn S, Kunentrasai N, et al. Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 1997; 176:313-21. 8. Hommel D, Talarmin A, Deubel V, Reynes JM, Drouet MT, Sarthou JL, et al. Dengue encephalitis in French Guiana. Res Virol. 1998; 149:235-8. 9. Murgue B, Deparis X, Chungue E, Cassar O, Roche C. Dengue: an evaluation of dengue severity in French Polynesia based on an analysis of 403 laboratory confirmed cases. Trop Med Int Health 1999; 4:765-73. 10. Pan American Health Organization. Dengue hemorrhagic fever in Venezuela. Epidemiol Bull 1990; 11: 7-9. 11. Calvert AE, Huang CYH, Kinney RM, Roehrig T. Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge. J Gen Virol 2006; 87:339-46. 12. Wengler G. The carboxy-terminal part of NS3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 1991; 184: 707-15. 13. Lindenbach BD, Rice CM. trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 1997; 71:9608-17. 14. Roehrig JT. Antigenic structure of flavivirus protein. Adv Virus Res 2003; 59:141-75. 15. Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD. High level expression of recombinant dengue viral NS1 protein and its potential use as a diagnostic antigen. J Med Virol 2001; 65:553-60. 16. Anderson NG, Anderson NL. Twenty years of two- dimensional electrophoresis: past, present and future. Electrophoresis 1996; 17: 443-53. 17. Edman P. A method for the determination of the amino acid sequence of peptides. Arch Biochem Biophys 1949; 22: 475-83. 18. Corthals GL, Wasinger VC, Hochstrasser DF, Sanchez JC. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000; 21: 1104-15. 19. Berkelman T, Stenstedt T. 2-D Electrophoresis Principles and Methods, 1998. 20. Shuib AS, Chua CT, Hashim OH. Sera of IgA Nephropathy Patients contain a Heterogeneous Population of Relatively Cationic Alpha-heavy chains. Nephron 1998; 78:290-5. 21. Wengler G. The carboxy-terminal part of NS3 protein of the west Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 1991; 184: 707-15. 22. Anderson L, Seilhamer J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 1997; 18: 533-7. 23. Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. J Clin Microbiol 2000; 38:1053-7. 24. Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD. High level expression of recombinant dengue viral NS1 protein and its potential use as a diagnostic antogen. J Med Virol 2001; 65: 553-60. Universa Medicina Vol.26 No.2