This is a revised application that has incorporated all of the suggestions of the initial review and the second review. The reviewers were pleased that we incorporated all of the suggestions of the prior review in the first revised application. In the subsequent revision, new concerns were raised that included: 1) showing that actual infection increased expression by transcriptional mechanisms; 2) using more mature cells like monocytes, alveolar macrophages, and differentiated THP-1 cells; and preliminary data for the adenovirus studies. All of these concerns are addressed in the current application with preliminary data and new proposed studies. We show clearly that actual infection increases transcription of the IL-1 and IL-1ra genes, that we can infect and use alveolar macrophages and monocytes for these studies (we previously published studies showing that we could infect differentiated THP-1 cells; J. Clin. Invest. 93:474, 1994); and we show strong preliminary data to suggest that the adenovirus studies will be successful. Suggestions were also made to show that viral responsive enhancer elements could transfer the same responsiveness to basal promoters. In our prior studies, we showed that the NFIL-1beta/a site in the IL-1 promoter responded to the CMV IE genes. In the revised application, we show construction of a plasmid containing only a basal promoter and a NFIL-1betaA enhancer. The enhancer element conferred responsiveness to the CMV IE genes. These types of studies have been incorporated in the revised application. In summary, we have demonstrated that transcription is relevant to these studies and that the studies reflect what happens in relevant cells. There is now ample preliminary data to show that the studies will be successful. A major strength of the application is that we will be able to show how two different latent virus upregulate expression of the IL-1 and IL-1ra genes. Not only will the virus-responsive sequences in the genes be identified but we will also define areas in the viral proteins that are necessary to mediate their effects. This is now a very complete application, thanks to the prior reviews. We also feel that the results of these studies will enhance our understanding of how latent viruses cause lung disease. Thus, we hope that the reviewers will give the application sufficient priority so that it is funded.
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