Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
Correspondence
Sazaly AbuBakar
sazaly{at}um.edu.my
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ABSTRACT |
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MAIN TEXT |
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All DENV genes used in the present study were cloned into pEGFP-N1 expression vector (Clontech) as EGFP fusion protein, as previously described (Shafee & AbuBakar, 1999). The NS3 protease gene was cloned as a full-length NS3-EGFP, whereas the NS2B-NS3 construct was recloned from the original pQE-30 into pEGFP-N1 and designated NS2B-NS3185(pro). The construct contained 45 amino acids of the hydrophilic domain of NS2B and 185 amino acids from the amino terminus of NS3 (Yusof et al., 2000
; Clum et al., 1997
). Site-directed mutagenesis of NS3 and NS2B-NS3185(pro) was performed based on the earlier report of Clum et al. (1997)
. Histidine (His51), found within the catalytic triad of NS3 in both NS3 and NS2B-NS3185(pro), was replaced with Ala, hence inactivating the protein's proteolytic activities (Falgout et al., 1993
; Clum et al., 1997
; Yusof et al., 2000
). For transfection studies, cells were transfected with purified recombinant pEGFP-N1 using Tfx-20 transfection reagent, following the protocol provided by the manufacturer (Promega). Successful expression of the cloned DENV-2 genes was detected by examining the cell cultures under an inverted UV-microscope (Zeiss Axiovert 25). At 48 h after transfection, cells were fixed, counterstained with propidium iodide, and viewed using a confocal microscope (Leica TCS SP2). The presence of apoptotic cells in the cell cultures was detected using the Dead-End Colorimetric Apoptosis Detection System, following the protocol described by the manufacturer (Promega). The percentages of apoptotic cells and cells expressing EGFP were determined as previously detailed (Shafee & AbuBakar, 2002
).
Fluorescence and confocal microscopic analyses of the transfected cells showed that cells expressing prM-EGFP, truncated E [E34253-EGFP; NB-E-EGFP and E281423-EGFP; B-E-EGFP), NS1-EGFP, NS2A-EGFP (data not shown), NS3-EGFP, and cells expressing mNS3-EGFP and mNS2B-NS3185(pro)-EGFP] had similar morphology to cells expressing EGFP alone (Fig. 1). In general, the transfected cells showed diffuse fluorescence with some speckling in the cytoplasm; but intense fluorescing clusters around the nuclear periphery were noted in some cells expressing NS3-EGFP (Fig. 1
). Cells expressing NS2B-NS3185(pro)-EGFP, on the other hand, had highly vacuolated cytoplasm with NS2B-NS3185(pro)-EGFP noted within the vacuoles (Fig. 1
, thin arrow, arrowhead). Fluorescent membrane-bound vesicles were also noted in the culture medium of cells expressing NS2B-NS3185(pro)-EGFP (Fig. 1
, asterisk, thick arrow). Similar vesicular structures, however, were not noted in cells expressing mNS2B-NS3185(pro)-EGFP. Immunoblot analysis of the culture medium of NS2B-NS3185(pro)-EGFP-expressing cells sedimented at 20 000 g was performed using polyclonal antibodies raised against NS2B-NS3185(pro)-EGFP. A polypeptide band of
65 kDa was detected (Fig. 2
a, arrow). The size of this polypeptide band corresponded to the theoretical mass of NS2B-NS3185(pro)-EGFP. In addition, a protein band of
55 kDa (Fig. 2a
, asterisk) which corresponded to the expected size of NS3185(pro)-EGFP, was also detected, which suggested that the expressed NS2B-NS3185(pro)-EGFP was cleaved in vivo to release NS2B. The origin of a thin band below the suspected NS3185(pro)-EGFP band is unknown.
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In the present study, it was also noted that the percentage apoptosis for the NS2B-NS3185(pro)-EGFP-expressing cells was directly correlated (r=0·72, P<0·001, Pearson) with the number of cells showing the presence of the cytoplasmic vesicular structures. These vesicular structures were absent in cells expressing mNS2B-NS3185(pro)-EGFP (Fig. 1), implying that an active protease catalytic site of NS2B-NS3185(pro)-EGFP somehow contributed towards the formation of the vesicular structures. Since spontaneous cytoplasmic vacuolation by itself has never been reported to induce apoptosis, it is likely then the DENV-2 NS3 protease activity is responsible for it. It is proposed here that NS2B interacts directly with NS3 within the vesicular complexes to enhance the NS3 proteolytic activity in manner similar to that previously reported in other systems (Brinkworth et al., 1999
; Chambers et al., 1993
; Arias et al., 1993
; Jan et al., 1995
). Furthermore, since the vesicular complexes were also absent in cells expressing NS3-EGFP, it raised the possibility that the interaction between NS3 and its cofactor NS2B directly contributed towards the formation of the vesicular structures and accumulation of NS2B-NS3185(pro)-EGFP within these structures leads to the induction of apoptosis.
The importance of DENV NS3 protease in the induction of apoptosis is in line with results for a number of other virus-encoded proteases (Prikhod'ko et al., 2002; Li et al., 2002
; Goldstaub et al., 2000
; Barco et al., 2000
). In Langat virus infection, apoptosis was suggested to be mediated by direct binding of the viral NS3 to caspase-8 (Prikhod'ko et al., 2002
). Caspase-mediated pathways have also been suggested for the 3C protease-induced apoptosis of Enterovirus 71 and Poliovirus (Li et al., 2002
; Barco et al., 2000
). Whether the caspase pathways are also activated by DENV-2 infection is not presently known. However, based on our findings, it is suggested that in DENV-2 infection accumulation of the intracellular NS3 resulted in cleavage of the apoptosis initiator molecules that in turn triggers activation of the apoptotic pathways. This is possible since DENV NS3 has been shown to act proteolytically in cis and also in trans on a number of potential substrates (Kolykhalov et al., 1994
; Bartenschlager et al., 1995
; Yusof et al., 2000
) and, similar to the findings presented here, NS2B also appeared to function as a cofactor to the DENV-2 NS3 protease activity. Furthermore, alignment of the DENV-2 NS3 amino acid sequence with a number of known serine proteases antagonistic to the inhibitor-of-apoptosis proteins (IAPs) revealed the presence of a motif resembling the IAP-binding motif within the N terminus of NS3 (Fig. 2c
). The first Ala residue of the conserved IAP antagonist N-terminal sequence, however, is substituted with Glu in NS3. Whether this substitution affects the function of DENV-2 NS3 as a bona fide IAP binding protein, requires further investigation. Nonetheless, it is envisaged that DENV-2 NS3 may antagonize the inhibitory function of the IAPs, leading to increased activation of the initiator caspases such as caspase-9 or the downstream effector caspases such as caspase-3 and -7, similar to that mediated by serine proteases Smac/DIABLO or Omi/HtrA2 (Du et al., 2000
; Verhagen et al., 2000
; Srinivasula et al., 2000
, 2001
; Verhagen et al., 2002
).
The induction of apoptosis in cells expressing other DENV-2 genes was probably mediated by a non-specific mechanism; as the percentages of apoptotic cells observed in these cells were similar. A much lower percentage of apoptosis, approximately half that caused by nonspecific DENV-2 gene expression, however, was obtained in cells expressing EGFP alone. This suggests that the percentage of apoptosis noted in cells expressing DENV-2 proteins other than NS3 or NS2B-NS3185(pro) was probably due to the additive effects of DENV-2 proteins and EGFP accumulation. Intracellular accumulation of the DENV-2 proteins including the mutant forms of NS3 and NS2B-NS3185(pro) triggered apoptosis, perhaps by inducing the endoplasmic reticulum stress-induced apoptotic pathways as previously suggested (Despres et al., 1996).
In summary, results obtained from the present study showed that DENV-2 serine protease NS3 and its precursor protein NS2B-NS3185(pro) induced apoptosis in transiently transfected Vero cells. A significantly higher percentage of apoptosis was observed in cells expressing NS2B-NS3185(pro), suggesting the importance of NS2B as a cofactor for NS3-induced apoptosis. We have also shown that mutation of the NS3 proteolytic site reduced the capacity of NS3 to induce apoptosis. These findings support the importance of NS3 protease in DENV-2-induced apoptosis.
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ACKNOWLEDGEMENTS |
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Received 2 December 2002;
accepted 4 April 2003.
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