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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 19, 2003 as doi:10.1096/fj.02-0508fje. |
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Departments of
* Experimental Medicine and Biochemical Science and
Biology, University of Rome "Tor Vergata", Rome, Italy;
Institute of Biomedical Science, University of Chieti "G. D’Annunzio", Chieti, Italy;
INeMM, CNR, Rome, Italy; and
|| Institute of Microbiology, University of Rome "La Sapienza", Rome, Italy
2Correspondence: Institute of Microbiology, Faculty of Pharmacy, University of Rome "La Sapienza", Piazzale Aldo Moro, 5, 00185 Rome, Italy. E-mail: annateresa.palamara{at}uniroma1.it.
SPECIFIC AIMS
Although the replication strategies of influenza virus have been well defined, little is known about the cellular, biochemical pathways that support its replication. The objective of the present study was to elucidate the role of the intracellular redox state in controlling the life cycle of the influenza A virus. In particular, we studied: 1) the role of intracellular levels of reduced glutathione (GSH) and Bcl-2 expression in determining the permissivity of different cell lines to influenza A virus infection and 2) the mechanism(s) responsible for GSH/Bcl-2-induced modulation of viral replication.
PRINCIPAL FINDINGS
1. Bcl-2 expression affects intracellular GSH content and influenza A virus replication
To investigate the hypothesis that Bcl-2 expression influences host-cell permissivity to influenza viral replication by modulating the intracellular redox state, we used various cell lines characterized by different levels of Bcl-2 expression and GSH (Fig. 1
A–C):
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—Madin Darby canine kidney (MDCK) cells (highly permissive to influenza virus replication) transfected with a vector expressing the human bcl-2 gene (MDCKBcl-2 cells) or with a control vector (MDCK controls);
—NCI-H292 (human pulmonary cell line), which resembles MDCK cells in their high permissivity to influenza virus replication and nonexpression of Bcl-2; and
—SH-SY5Y (neuroblastoma) and U937 (promonocytic) cell lines, which constitutively express Bcl-2.
The intracellular GSH content was measured in each of these cell lines 24 h after infection (p.i.) with influenza virus A/PR8 virus or mock-infection.
After mock-infection, GSH concentrations were significantly higher in the Bcl-2-expressing cells than in those that do not express this protein. Compared with those of control MDCK cells (3.28±0.30 nmol/mg protein), significantly higher GSH levels were found in U937 cells (59.63±5.53 nmol/mg protein; P<0.001), SH-SY5Y cells (18.51±1.72 nmol/mg protein; P<0.0001), and MDCKBcl-2 cells (5.70±0.40 nmol/mg protein; P<0.005). The level of the antioxidant measured in NCI-H292 cells (6.46±0.98 nmol/mg protein) was also approximately twice as high as that of control MDCK cells, although these cells did not express Bcl-2 (P<0.001). In PR8-infected cell lines, the relative concentrations of GSH mirrored those of the mock-infected cells, although significant decreases were documented 24 h p.i. in both of the permissive, non-Bcl-2-expressing lines (MDCK and NCI-H292).
In an attempt to correlate these findings with PR8 replication, hemagglutinating activity in culture supernatants (an index of the production of mature virions) was measured at different time points after virus challenge. Viral replication in each of the cell lines was related to Bcl-2 expression and GSH levels. In particular, in NCI-H292 cells, which contained higher levels of GSH than MDCK cells, although they did not express Bcl-2, viral replication occurred, but the virus titers in the supernatant were significantly lower (–43.9% at 24 h p.i. and -48% at 48 and 72 h) than those of MDCK cells (P<0.05). The Bcl-2-expressing neuronal and monocytoid cells, whose GSH levels were much higher than those of the MDCK line, were still susceptible to PR8 infection but much less permissive to viral replication. Indeed, in SH-SY5Y and U937 cells, detectable virus titers were found 18 and 24 h p.i., respectively. However, at 72 h p.i., these titers were virtually unchanged, reflecting reductions in replication with respect to MDCK cells ranging from 98.5% ± 0.20 to 99.3% ± 0.05. Finally, compared with that observed in MDCK cells, PR8 replication in MDCKBcl-2 cells was also significantly (P<0.01) reduced. Tissue culture infectious dose (50%) assays of the supernatants of the different cell lines demonstrated that PR8 infectivity was not significantly impaired by Bcl-2 or GSH. Overall, these results suggest that the expression of Bcl-2 affects PR8 replication and the intracellular GSH content.
2. Two key steps of influenza virus life cycle are affected by Bcl-2 and GSH
In a second group of experiments, we attempted to identify the mechanisms underlying the inhibitory effects of Bcl-2 and GSH on PR8 replication. Western blot analysis revealed that expression of viral proteins, particularly the late-stage proteins, hemagglutinin (HA) and matrix (M1), was decreased (with respect to MDCK cells) in all cell lines containing high GSH/Bcl-2 levels. However, this decrease did not completely account for the diminished release of virus into the supernatant, suggesting that inhibition of viral protein synthesis is not the sole mechanism underlying the Bcl-2/GSH-mediated decrease in viral replication. Immunofluorescence analysis of the PR8 nucleoprotein (NP) showed that in permissive, Bcl-2 negative MDCK and NCI-H292 cells, NP was located predominantly in the nuclei 8 h p.i., but later (24 h p.i.), large amounts were found in the cytoplasm. In contrast, nuclear-cytoplasmic translocation of this protein appeared to be inhibited in cells expressing Bcl-2: At 24 h p.i., most of the MDCKBcl-2 cells displayed only small amounts of NP in the cytoplasm, and in SH-SY5Y cells, the NP was almost completely confined to the nucleus (Fig. 1B, a, c
). These data suggested that Bcl-2 may interfere with the nuclear export of viral ribonucleoproteins (vRNPs).
Finally, to better define the roles of GSH and/or Bcl-2 in the control of PR8 replication, viral protein expression and NP export were re-evaluated in Bcl-2-expressing cells in which GSH neosynthesis had been inhibited by BSO. MDCKBcl-2 and SH-SY5Y cells were pretreated with 1 mM BSO for 18 h prior to infection with PR8 and 24 h thereafter. As shown in Figure 1A
, inhibition of GSH synthesis enhanced the expression of viral proteins, especially HA and M1, but the nuclear retention of NP described above was completely (SH-SY5Y) or partially (MDCKBcl-2) unaffected by BSO treatment (Fig. 1B, b, d
).
Viral titers in the supernatant of these cells were related to the nuclear NP localization. Indeed, in the supernatant of BSO-treated MDCKBcl-2 cells, 1.3- to 2-fold increases in HA titers, with respect to untreated cells, were consistently observed in all experiments performed, whereas virus release from SH-SY5Y cells was unaffected by BSO (Fig. 1A
, boxes on the gels).
CONCLUSIONS AND SIGNIFICANCE
This study demonstrates that the antiapoptotic protein Bcl-2, which also exerts antioxidant effects, and GSH, a well-established antioxidant, are important modulators of the life cycle of influenza A virus, although they seem to operate at different levels. Constitutive and enforced expression of Bcl-2 was associated with higher GSH levels than those found in cells that do not express this protein, and replication of influenza virus was found to be inversely correlated with Bcl-2 expression and GSH content. The results summarized in Figure 2
indicate that Bcl-2 and GSH affect at least two key steps in the influenza virus life cycle: expression of late viral proteins and translocation of vRNPs from the nucleus to the cytoplasm. The former effect seems to be GSH-dependent, and the latter appears to be mediated by Bcl-2.
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The role of the intracellular redox state in the control of viral replication and pathogenesis has been well documented. Studies by our group have demonstrated that redox alterations during viral infection depend on GSH depletions, which vary in intensity, duration, and mechanism of induction depending on the type of virus and the host-cell infected. Furthermore, many antioxidant substances have been shown to inhibit the replication of different types of viruses in vivo and in vitro.
Our finding of high GSH levels in Bcl-2-expressing cells confirms previous observations in different experimental systems. The reducing conditions produced by high GSH levels might conceivably interfere with disulfide-bond formation in the PR8 HA molecule, thus impeding its correct folding and insertion in the cell membrane. This phenomenon could explain, at least in part, the reduced quantities of virus released into the supernatant of infected, GSH-rich cells. Bcl-2 also seems to exert a direct (i.e., non-GSH-mediated), inhibitory effect on PR8 replication. Our findings indicate that this protein, which is anchored to the nuclear membrane, interferes with nuclear export of vRNPs. To our knowledge, our findings are the first evidence of a dual (i.e., GSH-mediated, Bcl-2-mediated) mechanism underlying redox-dependent modulation of viral replication.
Several reports have stressed the importance of Bcl-2 in blocking virus-induced apoptosis, and this effect has been associated with the persistence of normally cytopathic viruses within cells. Bcl-2 is known to be highly expressed in mouse brain and human adult neocortex, and its presence in cultured neurons has been correlated with resistance to Sindbis virus-induced cell death and the establishment of persistent viral infection. Previous reports indicate that influenza virus can reach the brain through the olfactory bulb, where it selectively targets several structures implicated in the pathogenesis of neuropsychiatric disturbances and behavioral changes. The mechanisms leading to these changes are still obscure. However, based on our findings, it is possible to hypothesize that the presence of Bcl-2 and GSH in cells of the central nervous system could down-regulate influenza virus replication without completely blocking it. This situation would result in a constant production of moderate amounts of infectious virions, which might favor the establishment of persistent infection of these cells. Studies are in progress in our laboratory to better define the mechanism(s) underlying this phenomenon.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0508fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0508fje 
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