Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The ␣,␤-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-␥, and tumor necrosis factor-␣ by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-B DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-B1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-B consensus sequence. Matrix-assisted laser desorption/ionization time-offlight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.Cigarette smoke produces profound suppression of pulmonary immunity, resulting in an increased incidence and severity of respiratory tract infections. A recent Institute of Medicine study concluded that smoking increased the incidence of influenza and bacterial pneumonia and accounted for 19,000 smoking-related deaths per year (1). Children infected with Mycobacterium tuberculosis are five times more likely to develop pulmonary tuberculosis if exposed to cigarette smoke (2), and smoking doubles the risk of developing Pneumocystis carinii pneumonia in human immunodeficiency virus-infected individuals (3). Several studies have demonstrated that smoking suppresses T and B cell responses in the lungs without affecting cells in the peripheral blood (4 -7), but little research has been done to elucidate the underlying mechanism behind this phenomenon.We have recently identified two classes of immunosuppressive compounds in cigarette smoke. The dihydroxyphenols (hydroquinone and catechol) in the particulate phase inhibit T cell proliferation by blocking cell cycle progression in late G 1 and S phase (8 -12). In addition, the ␣,␤-unsaturated aldehydes, acrolein (CH 2 ACHCHO) and crotonaldehyde (CH 3 CHACHCHO) in the gas phase of cigarette smoke inhibit the production of several proinflammatory cytokines including IL-2, 4 tumor necrosis factor-␣, and granulocyte-macrophage colony-stimulating factor, with an IC 50 of 3 and 6 M, respectively (13). The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) have IC 50 values of Ͼ1500 M. The typical cigarett...

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Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus that causes infections of the respiratory tract, urinary tract, and gastrointestinal tract. Assembly of virus particles requires condensation and encapsidation of the linear viral genome. This process requires sequence specific binding of two viral proteins, called IVa2 and L4-22K, to a conserved sequence located at the left end of the viral genome, called the packaging sequence (PS). IVa2 and an alternatively spliced form of L4-22K, called L4-33K, also function as transcriptional activators of the major late promoter (MLP), which encodes viral structural and core proteins. IVa2 and L4-33K bind to identical conserved DNA sequences downstream of the MLP, called the downstream element (DE), to activate transcription. To begin to dissect how the IVa2, L4-22K, and L4-33K proteins simultaneously function as transcriptional activators and DNA packaging proteins, we need to understand the thermodynamics of assembly of these proteins on DNA that contains the PS as well as the DE. Toward this end, we have characterized the self-assembly properties of highly purified, recombinant L4-22K protein. We show that L4-22K reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme. We show that the smallest polymerizing unit is likely the L4-22K monomer (s(20,w) = 2.16 ± 0.04 S) and that the monomer assembles with itself and/or other aggregates with an equilibrium association constant, L, of 112 (102, 124) μM(-1) (0.1 M NaCl, pH 7, 25 °C). A mechanistic consequence of an isodesmic, indefinite assembly process is that the free concentration of the smallest polymerizing unit cannot exceed 1/L. We discuss the implications of this observation with respect to the thermodynamics of assembly of L4-22K and IVa2 on the PS.

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.