Journal of General Virology (1992), 73, 2999-3003.
Printed in Great Britain
2999
Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies R. Bulich and J. G. Aaskov* Centre for Molecular Biotechnology, Queensland University of Technology, George Street, Brisbane, Australia
Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-
shocked cells, but were first detected in infected cells approximately 32 h post-infection. P E P S C A N epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (gRNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (eKKAR°) and spans a possible second site for the initiation of synthesis of core protein (laPFN~MLKR~e).
Dengue viruses, members of the family Flaviviridae, are a major cause of morbidity and mortality in tropical countries of the world (Halstead, 1988). Although there is no direct evidence for the involvement of the cell nucleus in flavivirus replication, virusdirected RNA and protein synthesis do occur in the perinuclear region of infected cells (Westaway & Ng, 1980; Ng et al., 1983), and proteins from a number of R N A viruses, including the core protein of dengue 4 virus, have been reported to localize to the nucleus of infected cells (Anzai & Ozaki, 1969; Jackson et al., 1982; Young et al., 1987; Buckley & Gould, 1988; Tadano et al., 1989). However, none of the epitopes recognized by the antibodies used in these studies was defined, raising the possibility of serological cross-reactions between viral epitopes and those of normal cellular proteins (Gould et al., 1983). Standard protocols (Zola, 1987) were used to derive six IgG1 monoclonal antibodies (MAbs) from hybridomas resulting from the fusion of spleen cells from dengue 2 virus core protein-immunized BALB/c mice and SP2/0-Ag14 myeloma cells. Dengue 2 virus core protein was obtained by mixing 25 ~tl sucrose gradient-purified dengue 2 virus (Aaskov et al., 1988) with prestained Mr markers (2 ktl, Bio-Rad) prior to PAGE (Laemmli, 1970), and then, after e!ectrophoresis, eluting protein from a 5 mm × 3 mm strip of acrylamide from immediately in front of the Mr 18 500 prestained Mr marker. The remainder of the gel was stained with Coomassie blue to confirm that the strip of acrylamide recovered contained only core protein.
In a PEPSCAN assay (Geysen et al., 1987), polyclonal sera from mice immunized with dengue 2 virus core protein reacted with many peptides but all six MAbs recognized octapeptides composed of amino acid sequences (Deubel et al., 1986) found in a similar region of the dengue 2 virus core protein (9RTNPFNMLKRE19) (Fig. 1). The only protein sequences in the GenBank database to have significant amino acid identity with this region were an insecticidal protein, ISRH-3, from Bacillus thuringiensis (127tTkFNtwKRE136) (Sen et al., 1988) and a DNA gyrase A subunit from Escherichia coli (661TeFNrL666) (Swanberg & Wang, 1987). Antibody pairs 6F3.1 and 6F3.2, 4D1 and 2H12, and 4H9.1 and 2E5.1 appeared to recognize similar epitopes, so only one of each pair was used in some subsequent assays. The MAbs also reacted with the core protein of sucrose gradient-purified dengue 2, dengue 4 and, albeit weakly, dengue 1 viruses (Fig. 2) in Western blots (Aaskov et al., 1988). There was no detectable reaction with dengue 3 virus. Core protein was first detected by indirect ELISA in acetone-fixed (4 °C, 1 min) cytospin (1000 r.p.m., 2 min) preparations of dengue 2 virus-infected C6/36 Aedes albopictus cells 32 h after infection. MAbs (mouse ascitic fluid diluted 1:20 in PBS pH 7-4) were added to cell monolayers for 45 min followed, after three washes in PBS, by horseradish peroxidase-labelled rabbit antimouse immunoglobulin (Dako). Following a further three washes in PBS, a substrate/chromogen solution of H2Oz/diaminobenzidine was added to the cell monolayers to localize antibody, and a counterstain of Mayer's
0001-1212 © 1992 SGM
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(a)
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Fig. 2. Reaction of MAbs with structural proteins of sucrose gradientpurified dengue 1, 2, 3 and 4 viruses (a to d) in Western blots,
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Fig. 1. Reaction of polyclonal mouse serum (a), non-immune mouse serum (b) and MAbs 6F3.1, 6F3.2, 4D1, 2H12, 4H9.1 and 2E5.1 (c to h) with octapeptides composed of overlapping amino acid sequences from the core protein of dengue 2 virus in a P E P S C A N assay. Peptides are numbered from the N terminus of the core protein. The amino acid sequences of the peptides recognized by the MAbs are shown.
haematoxylin was used to identify cell nuclei. At 32 h, antigen could be seen in the cytoplasm and perinuclear region, with an occasional cell showing some nuclear staining. By 72 h post-infection, approximately 70~ of cells contained core protein detectable with the MAbs• As well as the cytoplasmic and perinuclear staining seen earlier, the cells now contained intensely staining areas in or on the nucleus, but in no instance was staining confined to nucleoli (Fig. 3). Similar patterns of staining were seen in vertebrate cells (LLC-MKII, BHK) infected with dengue 2 virus, and similar but less intense staining was observed in dengue 4 virus-infected C6/36 cells. No staining was observed in C6/36 cells infected with dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus, or in cells heat-shocked at 41 °C for 4 h. Detection by using anti-dengue 2 virus core protein MAbs of antigen in or on the nucleus of dengue 4 virusinfected C6/36 cells confirms the previous reports of
Tadano et aL (1989), although the almost exclusive localization of core proteins to nucleoli was not observed. These results, and the amino acid sequence comparisons discussed above, would seem to exclude the possibility that our MAbs cross-reacted with constituents of host cells such as heat-shock proteins, which are produced in response to virus infection (Garry el al., 1983) or exposure to elevated temperatures (Tissieres et al., 1974) and which may localize to the nucleolus of stressed cells (Milarski & Morimoto, 1989). Although the determinant 16LKR TM appeared to be critical for the binding of MAbs 6F3.1 and 6F3.2, and 16LKRE19 for the remaining four antibodies, it is possible that adjacent amino acids or protein conformation may also contribute to the function of these epitopes. Predictions of secondary protein structure (Chou & Fasman, 1974; Parker et aL, 1986) suggest that the conformation of the dengue 2 virus core protein is more closely related to that of dengue 4 virus than to those of any of the other flaviviruses studied in the region recognized by our MAbs. This may explain the limited cross-reactivity of these MAbs despite dengue 1, 2, 3 and 4, Murray Valley encephalitis and Kunjin viruses sharing the amino acid sequence N M L K R in this region of the core protein (Mason et aL, 1987; Osatomi et al., 1988; Zhao et al., 1986; Cola et al., 1988; Dalgarno et aL, 1986)• Failure of the MAbs to recognize core protein in dengue 1 virus-infected cells when they had reacted with pure virus in Western blots (Fig. 2) may simply reflect the relative amounts of core protein present in each of the assays. The intracellular distribution of core protein detected with our MAbs contrasts with the generalized cytoplasmic staining observed when using the anti-envelope protein MAb IB7 (Henchal et al., 1985) (Fig. 3) and
Short communication
3001
C6/36
LLC-MK
BHK
Fig. 3. Indirect ELISA employing anti-core (6F3.1, 2H12 and 4H9.1) or anti-envelope protein (1B7) MAbs and dengue 2 virusinfected C6/36, BHK or LLC-MK cells. Panel (a) shows indirect ELISA employing MAb 6F3. I and dengue 4 virus-infected C6/36 cells. Arrows indicate localized concentrations of core protein.
suggests that some of the core proteins remain attached to or inserted in the nuclear m e m b r a n e rather than being incorporated into mature virions. To examine this possibility, C6/36 cells were disrupted in hypotonic Tris buffer containing deoxycholate and NP40 (Naeve & Trent, 1978) 72 h after infection with dengue 2 virus when nuclear localization of core protein was detectable in > / 7 0 ~ of cells. The resultant nuclei, cytoplasm and a dense m e m b r a n e aggregate were analysed by TricineS D S - P A G E (Schagger & v o n Jagow, 1987) followed by ELISA on Western blots (Aaskov et al., 1988). A band of core protein of similar Mr was detected in all preparations and in pure dengue 2 virus (Fig. 4). These data are consistent with the intracellular distribution of staining seen in indirect E L I S A with dengue 2 virus-infected cells (Fig. 3) and a flavivirus replication strategy in which core proteins are synthesized in the cytoplasm and then inserted, via their C termini, in the m e m b r a n e of the endoplasmic reticulum prior to assembly of mature virions and their release from the surface of infected cells (Coia et al., 1988). However, they do not indicate whether the core protein is inside the nucleus or embedded in the nuclear membrane. Previous reports of nuclear localization of flavivirus proteins also failed to demonstrate the presence of these proteins
1
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--46K --30K --22K --14K --6.5K --3.4K
Fig. 4. Indirect ELISA using MAb 6F3.1 to detect core protein in Western blots of cytoplasm (lanes 1 and 2), membrane aggregates (lanes 4 and 5) and nuclei (lanes 7 and 8) of dengue 2 virus-infected (lanes 2, 5 and 8) and uninfected (lanes 1, 4 and 7) C6/36 cells. Lane 3 contains pure dengue 2 virus and lane 7 pre-stained Mr markers.
inside the nucleus, although apparent staining of nucleoli is suggestive of internalization (Buckley & Gould, 1988 ; Tadano et al., 1989). The dengue 2 virus core protein contains three
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Short communication
putative nuclear localization signals. 6KKAR9 is the same as a signal found in the polyoma virus large T protein (28tKKAR 285) (Richardson et al., 1986), but would be absent from any core protein truncated at the second methionine residue (Met 15) from the N terminus of the full-length core protein (Castle et al., 1985; Osatomi et al., 1988). The second sequence (73KKSK76) resembles a nuclear localization sequence in nucleop l a s m i n ( 1 6 3 K K K K 1 6 6 ) (Burglin & De Robertis, 1987), and would be retained even if the core protein was truncated at Met 15 and the 12 or 13 amino acid hydrophilic region at the C terminus was removed prior to incorporation into mature virions (Speight & Westaway, 1989). Dengue 4 virus core protein contains neither of these motifs and yet it localizes to the nucleus of infected cells. Of potentially greater significance is the bipartite nuclear targeting sequence motif (Dingwall & Lasky, 1991) SSRKkeigrmlnilnRRRRl°°, variations of which are found in all flavivirus core proteins for which the derived amino acid sequence has been published. However, it remains to be demonstrated experimentally that these putative sequences are required for nuclear localization of dengue virus core proteins. The absence of more reports of nuclear localization of flavivirus core proteins may simply reflect the absence of appropriate reagents to detect these proteins. We wish to thank Dr Ed Westaway for helpful discussions about nuclear targeting motifs. This study was supported by a grant from the National Health and Medical Research Council.
References AASKOV, J. G., WILLIAMS,L , FLETCHER, J. & HAY, R. (1988). Failure of a dengue l sub-unit vaccine to protect mice against a lethal dengue virus infection. American Journal of Tropical Medicine and Hygiene 39,511 518. ANZAI, T. & OZAKI, Y. (1969). Intranuclear crystal formation of poliovirus: electron microscopic observations. Experimental and Molecular Pathology 10, 176-185. BUCKLEY, A. & GOULD, E. A. (1988). Detection of virus-specific antigen in the nuclei or nucleoli of cells infected with Zika or Langat virus. Journal of General Virology 69, 1913-1920. BURGLIN, T. R. & DE ROBERTIS, E. M. (1987). The nuclear migration signal of Xenopus laevis nucleoplasmin. EMBO Journal6, 2617 2625. CASTLE,E., NOWAK T., LEIDNER, U., WENGLER, G. & WENGLER,G. (1985). Sequence analysis of the viral core protein and the membrane associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology 145, 227-236. CHOU, P. Y. & FASMAN, G. D. (1974). Prediction of protein conformation. Biochemistry 13, 222-245. COIA, G., PARKER, M. D., SPEIGHT, G., BYRNE, M. E. & WESTAWAY, E. G. (1988). Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virusspecified proteins. Journal of General Virology 69, 1-21. DALGARNO, L., TRENT, D. W., STRAUSS, J. H. & RICE, C. i . (1986). Partial nucteotide sequence of the Murray Vatley encephalitis virus genome: comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins. Journal of Molecular Biology 187, 309-323.
DEUBEL, V., KINNEY, R. M. & TRENT, D. W. (1986). Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. Virology 155, 365-377. DINGWALL, C. & LASKY, R. A. (1991). Nuclear targeting sequences - a consensus. Trends in Biochemical Sciences 16, 478-481. GARRY, R. F., ULUG, E. T. & BOS~, H. R. (1983). Induction of stress proteins in Sindbis virus and vesicular stomatitis virus-infected cells. Virology 129, 319-332. GEYSEN, H. M., RODDA, S. J., MASON, T. J., TRIBBICK, G. & SCHOOFS, P. G. (1987). Strategies for epitope analysis using peptide synthesis. Journal of Immunological Methods 102, 259-274. GOULD, E. A., CHANAS, A. C., BUCKLEY, A. & CLEGG, C. S. (1983). Monoclonal immunoglobulin M antibody to JE virus that can react with a nuclear antigen in mammalian cells. Infection and Immunity 41, 774-779. HALSTEAD, S. B. (1988). Pathogenesis of dengue: challenges to molecular biology. Science 239, 476-481. HENCHAL., E. A., MCCOWN, J. M., BURKE, D. S., SEGUIN, M. C. & BRANDT, W. E. (1985). Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene 34, 162-169. JACKSON, D. A., CATON, A. J., McCgEADY, S. J. & COOK P. R. (1982). Influenza virus RNA is synthesized at fixed sites in the nucleus. Nature, London 296, 366-368. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680 685. MASON, P. W., MCADA, P. C., MASON,T. L. & FOURNIER, M. J. (1987). Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major non-structural protein NS1. Virology 161, 262 267. MILARSKI, K. L. & MORIMOTO, R. I. (1989). Mutational analysis of the human HSP70 protein. Distinct domains for nuclear localisation and adenosine triphosphate binding. Journal of Cell Biology 109, 1947-1962. NAEVE, C. W. & TRENT, D. W. (1978). Identification of Saint Louis encephalitis virus mRNA. Journal of Virology 25, 535-545. NG, M. L., PEDERSEN, J. S., TOH, B. H. & WESTAWAY, E. G. (1983). Immunofluorescent sites in Vero cells infected with the ftavivirus Kunjin. Archives of Virology 78, 177-190. OSATOMI, K., FUKE, I., TSURU, D., SHIBA,T., SAKAKI,Y. & SUMIYOSHI, H. (1988). Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins. Virus Genes 2, 99-108. PARKER, J. M. R., GUO, D. & HODGES, R. S. (1986). New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry 25, 5425-5432. RICHARDSON, W. D., ROBERTS, B. L. & SMITH, A. E. (1986). Nuclear location signals in polyoma virus large-T. Cell 44, 77 85. SCHAGGER, H. & YON JAGOW, G. (1987). Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry 166, 368-379. SEN, K., HONDA, G., KOYAMA,N., NISHtDA, M., NEKI, A., HIMENO, M. & KOMANO, T. (1988). Cloning and nucleotide sequences of the two 130 kDa insecticidal protein genes of Bacillus thuringiensis var. israelensis. Agricultural and Biological Chemistry 52, 873-878. SPEIGHT, G. & WESTAWAY,E. G. (1989). Carboxy-terminal analysis of nine proteins specified by the flavirus Kunjin: evidence that only the intracellular core protein is truncated. Journalof General Virology70, 2209-2214. SWANBERG,S. L. & WANe, J. C. (1987). Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase. Journal of Molecular Biology 197, 729-736. TADANO, M., MAKINO, Y., FUKUNAGA, T., OKUNO, Y. & FUKAI, K. (1989). Detection of dengue 4 virus core protein in the nucleus. I. A monoclonal antibody to dengue 4 virus reacts with the antigen in the nucleus and cytoplasm. Journal of General Virology 70, 1409-1415. TISSIERES, A., MITCHELL, H. K. & TRACY, U. M. (1974). Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. Journal of Molecular Biology 84, 389-398.
Short communication
WESTAWAY,E. G. & N6, M. L. (1980). Replication of flaviviruses: separation of membrane translation sites of Kunjin virus proteins and cell protein. Virology 106, 107-122. YOUNG, P. R., CHANAS,A. C., LEE, S. R., GOULD,E. A. & HOWARD, C. R. (1987). Localization of an arenavirus protein in the mlclei of infected cells. Journal of General Virology 68, 2465-2470. ZHAO,B., MACKOW,E., BUCKLER-WHITE,A., MARKOFF,L., CHANOCK,
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R. M., LAI, C.~J. & MAKINO,Y. (1986). Cloning full length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. Virology 155, 77-88. ZOLA, H. (1987). Monoclonal Antibodies: A Manual of Techniques. Boca Raton: CRC Press.
(Received 7 July 1992; Accepted 29 July 1992)
Printed in Great Britain
2999
Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies R. Bulich and J. G. Aaskov* Centre for Molecular Biotechnology, Queensland University of Technology, George Street, Brisbane, Australia
Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-
shocked cells, but were first detected in infected cells approximately 32 h post-infection. P E P S C A N epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (gRNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (eKKAR°) and spans a possible second site for the initiation of synthesis of core protein (laPFN~MLKR~e).
Dengue viruses, members of the family Flaviviridae, are a major cause of morbidity and mortality in tropical countries of the world (Halstead, 1988). Although there is no direct evidence for the involvement of the cell nucleus in flavivirus replication, virusdirected RNA and protein synthesis do occur in the perinuclear region of infected cells (Westaway & Ng, 1980; Ng et al., 1983), and proteins from a number of R N A viruses, including the core protein of dengue 4 virus, have been reported to localize to the nucleus of infected cells (Anzai & Ozaki, 1969; Jackson et al., 1982; Young et al., 1987; Buckley & Gould, 1988; Tadano et al., 1989). However, none of the epitopes recognized by the antibodies used in these studies was defined, raising the possibility of serological cross-reactions between viral epitopes and those of normal cellular proteins (Gould et al., 1983). Standard protocols (Zola, 1987) were used to derive six IgG1 monoclonal antibodies (MAbs) from hybridomas resulting from the fusion of spleen cells from dengue 2 virus core protein-immunized BALB/c mice and SP2/0-Ag14 myeloma cells. Dengue 2 virus core protein was obtained by mixing 25 ~tl sucrose gradient-purified dengue 2 virus (Aaskov et al., 1988) with prestained Mr markers (2 ktl, Bio-Rad) prior to PAGE (Laemmli, 1970), and then, after e!ectrophoresis, eluting protein from a 5 mm × 3 mm strip of acrylamide from immediately in front of the Mr 18 500 prestained Mr marker. The remainder of the gel was stained with Coomassie blue to confirm that the strip of acrylamide recovered contained only core protein.
In a PEPSCAN assay (Geysen et al., 1987), polyclonal sera from mice immunized with dengue 2 virus core protein reacted with many peptides but all six MAbs recognized octapeptides composed of amino acid sequences (Deubel et al., 1986) found in a similar region of the dengue 2 virus core protein (9RTNPFNMLKRE19) (Fig. 1). The only protein sequences in the GenBank database to have significant amino acid identity with this region were an insecticidal protein, ISRH-3, from Bacillus thuringiensis (127tTkFNtwKRE136) (Sen et al., 1988) and a DNA gyrase A subunit from Escherichia coli (661TeFNrL666) (Swanberg & Wang, 1987). Antibody pairs 6F3.1 and 6F3.2, 4D1 and 2H12, and 4H9.1 and 2E5.1 appeared to recognize similar epitopes, so only one of each pair was used in some subsequent assays. The MAbs also reacted with the core protein of sucrose gradient-purified dengue 2, dengue 4 and, albeit weakly, dengue 1 viruses (Fig. 2) in Western blots (Aaskov et al., 1988). There was no detectable reaction with dengue 3 virus. Core protein was first detected by indirect ELISA in acetone-fixed (4 °C, 1 min) cytospin (1000 r.p.m., 2 min) preparations of dengue 2 virus-infected C6/36 Aedes albopictus cells 32 h after infection. MAbs (mouse ascitic fluid diluted 1:20 in PBS pH 7-4) were added to cell monolayers for 45 min followed, after three washes in PBS, by horseradish peroxidase-labelled rabbit antimouse immunoglobulin (Dako). Following a further three washes in PBS, a substrate/chromogen solution of H2Oz/diaminobenzidine was added to the cell monolayers to localize antibody, and a counterstain of Mayer's
0001-1212 © 1992 SGM
3000
Short communication
(a)
25I20 I1, 2.5 "~2.0
(c)
n
'~" 1.0
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(d)
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Fig. 2. Reaction of MAbs with structural proteins of sucrose gradientpurified dengue 1, 2, 3 and 4 viruses (a to d) in Western blots,
l0 TNPFNMLK 11 NPFNMLKR 12 PFNMLKRE
.......1[ 1,.,,0,h,lhlSlldlhldhh,l,h,i.lh.,.1111...,lrllllln,l,.,ll[][[llll II................ I ................................................................................. I.......
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Fig. 1. Reaction of polyclonal mouse serum (a), non-immune mouse serum (b) and MAbs 6F3.1, 6F3.2, 4D1, 2H12, 4H9.1 and 2E5.1 (c to h) with octapeptides composed of overlapping amino acid sequences from the core protein of dengue 2 virus in a P E P S C A N assay. Peptides are numbered from the N terminus of the core protein. The amino acid sequences of the peptides recognized by the MAbs are shown.
haematoxylin was used to identify cell nuclei. At 32 h, antigen could be seen in the cytoplasm and perinuclear region, with an occasional cell showing some nuclear staining. By 72 h post-infection, approximately 70~ of cells contained core protein detectable with the MAbs• As well as the cytoplasmic and perinuclear staining seen earlier, the cells now contained intensely staining areas in or on the nucleus, but in no instance was staining confined to nucleoli (Fig. 3). Similar patterns of staining were seen in vertebrate cells (LLC-MKII, BHK) infected with dengue 2 virus, and similar but less intense staining was observed in dengue 4 virus-infected C6/36 cells. No staining was observed in C6/36 cells infected with dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus, or in cells heat-shocked at 41 °C for 4 h. Detection by using anti-dengue 2 virus core protein MAbs of antigen in or on the nucleus of dengue 4 virusinfected C6/36 cells confirms the previous reports of
Tadano et aL (1989), although the almost exclusive localization of core proteins to nucleoli was not observed. These results, and the amino acid sequence comparisons discussed above, would seem to exclude the possibility that our MAbs cross-reacted with constituents of host cells such as heat-shock proteins, which are produced in response to virus infection (Garry el al., 1983) or exposure to elevated temperatures (Tissieres et al., 1974) and which may localize to the nucleolus of stressed cells (Milarski & Morimoto, 1989). Although the determinant 16LKR TM appeared to be critical for the binding of MAbs 6F3.1 and 6F3.2, and 16LKRE19 for the remaining four antibodies, it is possible that adjacent amino acids or protein conformation may also contribute to the function of these epitopes. Predictions of secondary protein structure (Chou & Fasman, 1974; Parker et aL, 1986) suggest that the conformation of the dengue 2 virus core protein is more closely related to that of dengue 4 virus than to those of any of the other flaviviruses studied in the region recognized by our MAbs. This may explain the limited cross-reactivity of these MAbs despite dengue 1, 2, 3 and 4, Murray Valley encephalitis and Kunjin viruses sharing the amino acid sequence N M L K R in this region of the core protein (Mason et aL, 1987; Osatomi et al., 1988; Zhao et al., 1986; Cola et al., 1988; Dalgarno et aL, 1986)• Failure of the MAbs to recognize core protein in dengue 1 virus-infected cells when they had reacted with pure virus in Western blots (Fig. 2) may simply reflect the relative amounts of core protein present in each of the assays. The intracellular distribution of core protein detected with our MAbs contrasts with the generalized cytoplasmic staining observed when using the anti-envelope protein MAb IB7 (Henchal et al., 1985) (Fig. 3) and
Short communication
3001
C6/36
LLC-MK
BHK
Fig. 3. Indirect ELISA employing anti-core (6F3.1, 2H12 and 4H9.1) or anti-envelope protein (1B7) MAbs and dengue 2 virusinfected C6/36, BHK or LLC-MK cells. Panel (a) shows indirect ELISA employing MAb 6F3. I and dengue 4 virus-infected C6/36 cells. Arrows indicate localized concentrations of core protein.
suggests that some of the core proteins remain attached to or inserted in the nuclear m e m b r a n e rather than being incorporated into mature virions. To examine this possibility, C6/36 cells were disrupted in hypotonic Tris buffer containing deoxycholate and NP40 (Naeve & Trent, 1978) 72 h after infection with dengue 2 virus when nuclear localization of core protein was detectable in > / 7 0 ~ of cells. The resultant nuclei, cytoplasm and a dense m e m b r a n e aggregate were analysed by TricineS D S - P A G E (Schagger & v o n Jagow, 1987) followed by ELISA on Western blots (Aaskov et al., 1988). A band of core protein of similar Mr was detected in all preparations and in pure dengue 2 virus (Fig. 4). These data are consistent with the intracellular distribution of staining seen in indirect E L I S A with dengue 2 virus-infected cells (Fig. 3) and a flavivirus replication strategy in which core proteins are synthesized in the cytoplasm and then inserted, via their C termini, in the m e m b r a n e of the endoplasmic reticulum prior to assembly of mature virions and their release from the surface of infected cells (Coia et al., 1988). However, they do not indicate whether the core protein is inside the nucleus or embedded in the nuclear membrane. Previous reports of nuclear localization of flavivirus proteins also failed to demonstrate the presence of these proteins
1
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5
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8
--46K --30K --22K --14K --6.5K --3.4K
Fig. 4. Indirect ELISA using MAb 6F3.1 to detect core protein in Western blots of cytoplasm (lanes 1 and 2), membrane aggregates (lanes 4 and 5) and nuclei (lanes 7 and 8) of dengue 2 virus-infected (lanes 2, 5 and 8) and uninfected (lanes 1, 4 and 7) C6/36 cells. Lane 3 contains pure dengue 2 virus and lane 7 pre-stained Mr markers.
inside the nucleus, although apparent staining of nucleoli is suggestive of internalization (Buckley & Gould, 1988 ; Tadano et al., 1989). The dengue 2 virus core protein contains three
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putative nuclear localization signals. 6KKAR9 is the same as a signal found in the polyoma virus large T protein (28tKKAR 285) (Richardson et al., 1986), but would be absent from any core protein truncated at the second methionine residue (Met 15) from the N terminus of the full-length core protein (Castle et al., 1985; Osatomi et al., 1988). The second sequence (73KKSK76) resembles a nuclear localization sequence in nucleop l a s m i n ( 1 6 3 K K K K 1 6 6 ) (Burglin & De Robertis, 1987), and would be retained even if the core protein was truncated at Met 15 and the 12 or 13 amino acid hydrophilic region at the C terminus was removed prior to incorporation into mature virions (Speight & Westaway, 1989). Dengue 4 virus core protein contains neither of these motifs and yet it localizes to the nucleus of infected cells. Of potentially greater significance is the bipartite nuclear targeting sequence motif (Dingwall & Lasky, 1991) SSRKkeigrmlnilnRRRRl°°, variations of which are found in all flavivirus core proteins for which the derived amino acid sequence has been published. However, it remains to be demonstrated experimentally that these putative sequences are required for nuclear localization of dengue virus core proteins. The absence of more reports of nuclear localization of flavivirus core proteins may simply reflect the absence of appropriate reagents to detect these proteins. We wish to thank Dr Ed Westaway for helpful discussions about nuclear targeting motifs. This study was supported by a grant from the National Health and Medical Research Council.
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(Received 7 July 1992; Accepted 29 July 1992)
