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J Virol, June 1998, p. 4849-4857, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Major Component of I
B
Proteolysis Occurs Independently
of the Proteasome Pathway in Respiratory Syncytial
Virus-Infected Pulmonary Epithelial Cells
Mohammad
Jamaluddin,1
Antonella
Casola,2
Roberto P.
Garofalo,2
Youqi
Han,1
Todd
Elliott,2
Pearay L.
Ogra,2 and
Allan R.
Brasier1,*
Department of Medicine and Sealy Center for
Molecular Science1 and
Department of
Pediatrics,2 University of Texas Medical Branch,
Galveston, Texas 77555-1060
Received 8 October 1997/Accepted 24 February 1998
Previously we showed that infection of human type II airway
epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in
the cytoplasm by association with the I
B inhibitor and can be released only following I
B degradation. In this study, we examined the spectrum of I
B isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In
contrast to the rapid and robust activation of RelA DNA binding that
peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-
), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak
until 24 h after infection. A549 cells expressed the I
B inhibitory subunits I
B
, I
B
, and p105; however, following
either stimulus, only the I
B
and I
B
steady-state
levels declined in parallel with the increase in RelA DNA-binding
activity. The >120-min half-life of I
B
in control
cells was shortened to 5 min in TNF-
-stimulated cells and to 90 min
in pRSV-infected cells. Although I
B
was resynthesized within
30 min following recombinant human TNF
treatment due to a robust
25-fold increase of I
B
mRNA expression (the RelA:I
B
positive feedback loop), following pRSV infection, there was no
reaccumulation of I
B
protein, as infected cells produced only a
3-fold increase in I
B
mRNA at 24 h, indicating the
RelA:I
B
positive feedback loop was insufficient to restore
control I
B
levels. I
B
proteolysis induced by TNF-
occurred through the 26S proteasome, as both 26S proteasome activity and I
B
proteolysis were blocked by specific inhibitors
lactacystin, MG-132, and ZLLF-CHO. Although total proteasome activity
in 24-h pRSV-infected lysates increased twofold, its activity was
>90% inhibited by the proteasome inhibitors; surprisingly, however, I
B
proteolysis was not. We conclude that RSV infection produces I
B
proteolysis through a mechanism primarily independent of the
proteasome pathway.
*
Corresponding author. Mailing address: Division of
Endocrinology, University of Texas Medical Branch, 301 University
Blvd., MRB 8.138, Galveston, TX 77555-1060. Phone: (409) 772-2824. Fax: (409) 772-8709. E-mail: arbrasie{at}utmb.edu.
J Virol, June 1998, p. 4849-4857, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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