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Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.The large tumor (T) antigen encoded by simian virus 40 (SV40) is a phosphoprotein that has diverse roles in viral DNA replication and transformation (for reviews, see references 7, 19, 43, and 58). The initiation of DNA replication is a series of tightly regulated events. At first, T antigen binds to the 64-bp core origin of replication in a sequence-specific manner. The two pairs of GAGGC pentanucleotides, termed site II, in the core origin serve as the major binding site for T antigen (16). In the presence of ATP, individual monomers of T antigen multimerize cooperatively into a double hexamer, a bilobed structure encircling the origin (12,28,63,67). Interestingly, preformed hexamers of T antigen were shown to be active in origin DNA binding and unwinding, implying that T antigen is versatile in performing these activities (62).Formation of double hexamers is well supported by either of the two assembly units made up of pentanucleotides 1, 3, and the early palindrome (EP) region or pentanucleotides 2, 4, and the AT track (23,24,52). An intact origin, however, is needed for efficient unwinding (13, 33). After hexamerization, T antigen partially melts the EP region and untwists the AT track (3,5,6,15,32). At the expense of ATP hydrolysis, the distorted origin is subsequently unwound bidirectionally by the helicase activity of T-antigen double hexamers (36,49,54). Once unwinding is initiated, the two hexamers likely remain associated with each other, with the separated single-stranded DNA threading through the hexameric channels (50, 67). At least three other host protei...

Endogenous glucocorticoid (GC) activation is regulated by the intracellular GC-activating and -inactivating enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)1 and 11β-HSD2, respectively, that catalyze interconversion of inert cortisone and its bioactive metabolite cortisol. Because endogenous GCs are critically implicated in suppressing the asthmatic state, this study examined the roles of the 11β-HSD enzymes in regulating GC activation and bronchoprotection during proasthmatic stimulation. Airway hyperresponsiveness to methacholine and inflammation were assessed in rabbits following inhalation of the proasthmatic/proinflammatory cytokine IL-13 with and without pretreatment with the 11β-HSD inhibitor carbenoxolone (CBX). Additionally, IL-13-induced changes in 11β-HSD isozyme expression and GC metabolism were examined in epithelium-intact and -denuded tracheal segments and peripheral lung tissues. Finally, the effects of pretreatment with CBX or 11β-HSD2-targeted siRNAs were investigated with respect to cortisol prevention of IL-13-induced airway constrictor hyperresponsiveness and eotaxin-3 production by airway epithelial cells. IL-13-exposed rabbits exhibited airway hyperresponsiveness, inflammation, and elevated bronchoalveolar lung fluid levels of eotaxin-3. These responses were inhibited by pretreatment with CBX, suggesting a permissive proasthmatic role for 11β-HSD2. Supporting this concept, extended studies demonstrated that 1) IL-13-treated tracheal epithelium and peripheral lung tissues exhibit upregulated 11β-HSD2 activity, 2) the latter impairs cortisone-induced cortisol accumulation and the ability of administered cortisol to prevent both IL-13-induced heightened airway contractility and eotaxin-3 release from epithelial cells, and 3) these proasthmatic responses are prevented by cortisol administration in the presence of 11β-HSD2 inhibition. Collectively, these data demonstrate that the proasthmatic effects of IL-13 are enabled by impaired endogenous GC activation in the lung that is attributed to upregulation of 11β-HSD2 in the pulmonary epithelium.

The immune-escape strategy employed by human oncogenic adenovirus type 12 (Ad12) involves downregulation of major histocompatibility complex class I (MHC-I) transcription by disabling the transactivator NF-B (p50/p65). This is accomplished by the Ad12 E1A protein (E1A-12), which prevents NF-B from becoming phosphorylated by the protein kinase A catalytic subunit (PKAc). In this study, we examined the interactions between E1A-12 and NF-B. Our data show that an E1A-12 mutant retaining the N-terminal 66 amino acids was as effective as the wild-type E1A-12 protein (266 amino acids) in binding p65, preventing phosphorylation of p65-Ser 276 , and inhibiting transactivation. In contrast, the nontumorigenic adenovirus type 5 E1A protein (E1A-5) and other E1A-12 mutants lacking the N-terminal regions were severely defective in these activities. Further studies revealed that an N-terminal peptide consisting of residues 1 to 40 of E1A-12 was able to associate directly with p65 in vitro and prevent PKAc from phosphorylating p65-Ser 276 . In the absence of the N terminus, there is an almost complete loss of E1A-12 binding to p65. These findings provide solid evidence for the role of the E1A-12 N terminus as an NF-B binding domain. Significantly, this study indicates that the E1A-12 N terminus prevents PKAc from gaining access to p65 to account for Ser 276 hypophosphorylation. The E1A-12 N terminus interaction with p65 serves as a key explanation of how Ad12 downregulates MHC-I transcription and contributes to oncogenesis by escaping cytotoxic T lymphocytes.

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