Viral infection leads to the transient expression of early inflammatory genes. The proteins encoded by these genes enhance recognition of the infected cells by the host immune system. A group of proteins, called interferons (IFNs), 1 can directly inhibit viral replication. Type I IFNs are encoded by a family of closely related ␣ genes and a single IFNB gene, which are all localized on chromosome 9 (1, 2). The sequences that regulate inducible transcription of these genes are localized within the 100-nucleotide 5Ј-end of the transcriptional start of IFNA and IFNB genes (3, 4). These regions contain a number of a short overlapping GAAAGT-rich sequences that serve as a binding sites for multiple transcriptional factors. Several elements that function as positive regulatory domains (PRD) in virus-infected cells were identified in the promoter region of the IFNB gene (5). It was further shown that the region designated as PRDII contains an NF-kB binding site (6 -8) to which the NF-kB heterodimers induced upon viral infection bind as well as the HMG protein (9). The PRDIV domain was shown to bind the ATF-2 factor and octamer binding protein (10), and the PRDI and PRDIII regions serve as high affinity sites for the binding of the interferon regulatory factors (IRF-1 and IRF-2) (6, 11, 12). The transcriptional activation of this promoter, in virus-infected cells, is a result of the interaction among these multiple transcriptional factors (13) where the virus-induced binding of p50⅐p65 heterodimer plays a crucial role (14,15). In contrast, the IFNA gene promoter region does not contain an NF-B binding site. However, both the murine and human IFNA virus-inducible regions (VRE) contain multiple repeats to the GAAAGT and AAGTGA elements that could serve as binding sites for the IRF-1 and IRF-2 factors (16 -18). In addition, the deletion and mutation analysis of the murine IFNA4 promoter region identified an essential element, named ␣F1 (19), which serves as a recognition site for DNA binding proteins p68 and p96 (20). In the human IFNA1 promoter region, another essential binding site interacting with the TG protein was identified (21). Recent studies have identified a distinct multisubunit complex in virus-induced cells (22).The presence of IRF-like binding sites in the promoter region of the IFNA and IFNB genes indicated that the IRF factors recognizing these sites play an essential role in the induction of IFN genes. The original results of Harada et al. (11) suggested that the up-regulation of these genes is mediated by IRF-1, while the closely related IRF-2 suppresses the expression of these genes. However, the essential role of IRF-1 and IRF-2 in the regulation of IFNA and IFNB gene expression in infected cells was disputed by the finding that mice containing the homozygous deletion of IRF-1 or IRF-2, or fibroblast derived from these mice, were able to induce IFNA and IFNB gene expression upon infection with Newcastle Disease virus (NDV) to the same level as the wild-type mice or cells (23, 24). IRF-1 was shown to pla...

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J.A., Gerlt, J.A., Kozarich, J.W., Koo, C.W., Shah, V.J., Kenyon, G.L., Neidhart, D.J., Fujita, J., & Petsko, G.A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R)- and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., Kenyon, G.L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.R., Petsko, G.A., & Kenyon, G.L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S)-atrolactate, with the exception that the side chain of His 297 is tilted and displaced approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S)-mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p-(bromomethyl)mandalate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pKa of His 297 in wild-type MR.

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Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the interferon regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the interferon-mediated transcriptional activation of the interferon-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-3. We further show that ectopic expression of vIRF-1 in NIH 3T3 cells confers resistance to tumor necrosis factor alpha-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the interferon consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 243. While no binding between the full-size IRF-3 and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 1623 to 2414) of the transcriptional coactivator p300 which could also bind IRF-3 and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p300.