The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.

HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.

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Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Our earlier reports demonstrated that Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk) and that these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk failed to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription was enhanced when Bright and Btk were coexpressed. In contrast, neither Bright nor Btk alone led to activation of heavy chain transcription. Furthermore, Bright function required both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright was not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitated with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk.Bright (B-cell regulator of immunoglobulin [Ig] heavy chain transcription) is a B-cell-restricted transcription factor that binds specific A-T-rich sequences. The protein consists of an acidic amino-terminal domain, a DNA-binding A-T-rich interaction domain, a putative transactivation domain, and a protein interaction domain (18). The carboxyl-terminal domain of Bright currently has no assigned function. Bright was originally identified in an antigen-specific B-cell line, BCg3R-1d, as a mobility-shifted complex induced after stimulation with interleukin-5 (IL-5) and antigen (54). Binding sites for Bright were originally identified 5Ј of the basal promoter of the V1 S107 gene but also exist within the matrix association regions on either side of the intronic enhancer (53, 55). Bright binding to the 5Ј-flanking sequences of the V1 S107 variable heavy chain (V H ) promoter correlated with two-to sixfold increases in heavy chain mRNA levels in response to 55). Deletion of Bright binding sites flanking the V1 promoter resulted in lack of antigen-and IL-5-stimulated heavy chain transcription (55). Bright expression is tightly regulated in normal murine lymphocytes, occurring in pre-B cells and late stages of B-cell differentiation (58). However, Bright is not present in detectable amounts in immature B cells, suggesting that it may not play a role in maintenance of Ig expression (58). On the other hand, Bright activity is induced in B cells activated in response to lipopolysaccharide (LPS), CD40 ligand stimulation, and anti-CD38 (55, 59). These data suggest that Bright enhances Ig heavy chain transcription above basal levels following B-cell activation.Our earlier results revealed that Bruton's tyrosine kinase (Btk) associates with Brig...

Human adenoviruses (HAdVs) are uniquely important “model organisms” as they have been used to elucidate fundamental biological processes, are recognized as complex pathogens, and are used as remedies for human health. As pathogens, HAdVs may effect asymptomatic or mild and severe symptomatic disease upon their infection of respiratory, ocular, gastrointestinal, and genitourinary systems. High-resolution genomic data have enhanced the understanding of HAdV epidemiology, with recombination recognized as an important and major pathway in the molecular evolution and genesis of emergent HAdV pathogens. To support this view and to actualize an algorithm for identifying, characterizing, and typing novel HAdVs, we determined the DNA sequence of 95 isolates from archives containing historically important pathogens and collections housing currently circulating strains to be sequenced. Of the 85 samples that were completely sequenced, 18 novel recombinants within species HAdV-B and D were identified. Two HAdV-D genomes were found to contain novel penton base and fiber genes with significant divergence from known molecular types. In this data set, we found additional isolates of HAdV-D53 and HAdV-D58, two novel genotypes recognized recently using genomics. This supports the thesis that novel HAdV genotypes are not limited to “one-time” appearances of the prototype but are of importance in HAdV epidemiology. These data underscore the significance of lateral genomic transfer in HAdV evolution and reinforce the potential public health impact of novel genotypes of HAdVs emerging in the population.

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.

Bright/ARID3a/Dril1, a member of the ARID family of transcription factors, is expressed in a highly regulated fashion in B lymphocytes, where it enhances immunoglobulin transcription three-to sixfold. Recent publications from our lab indicated that functional, but not kinase-inactive, Bruton's tyrosine kinase (Btk) is critical for Bright activity in an in vitro model system, yet Bright itself is not appreciably tyrosine phosphorylated. These data suggested that a third protein, and Btk substrate, must contribute to Bright-enhanced immunoglobulin transcription. The ubiquitously expressed transcription factor TFII-I was identified as a substrate for Btk several years ago. In this work, we show that TFII-I directly interacts with human Bright through amino acids in Bright's protein interaction domain and that specific tyrosine residues of TFII-I are essential for Bright-induced activity of an immunoglobulin reporter gene. Moreover, inhibition of TFII-I function in a B-cell line resulted in decreased heavy-chain transcript levels. These data suggest that Bright functions as a three-component protein complex in the immunoglobulin locus and tie together previous data indicating important roles for Btk and TFII-I in B lymphocytes.The transcription factor Bright (B-cell regulator of immunoglobulin H [IgH] transcription)/ARID3a/Dril1 is a B-cellspecific protein first discovered in a mature mouse B-cell line, BCg3R1-d, as a mobility-shifted protein complex that caused three-to sixfold increases in heavy-chain mRNA levels in response to stimulation with a T-dependent antigen and interleukin-5 (42, 43). Bright binds to AϩT-rich regions of the intronic heavy-chain enhancer previously identified as matrix association regions and to regions 5Ј of some variable heavychain (V H ) promoters, including the V1 S107 family gene (15,43). The cDNA for Bright was isolated in 1995, and the protein was shown to interact with DNA as a multimeric complex that included multiple copies of Bright (15). The Bright protein structure consists of an acidic N-terminal domain of unknown function, a DNA-binding AϩT-rich interaction domain (ARID), a putative transactivation domain, a protein interaction domain containing a helix-turn-helix region, and a small carboxyl-terminal domain with no assigned function.Earlier studies indicated that Bruton's tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease in both mice and humans, is a component of the Bright DNAbinding complex (28, 44). X-linked immunodeficiency disease in mice, or X-linked agammaglobulinemia (XLA) in humans, results in blocks in B-lymphocyte development that ultimately lead to a deficient production of serum antibodies (9,33,40). Patients with XLA are unable to fight normal bacterial infections without frequent intravenous Ig treatments. Although Btk was identified as the genetic defect in XLA many years ago, the mechanism by which Btk deficiencies lead to early blocks at the pro-B-to pre-B-lymphocyte stages in humans remains unclear.Recently, an in vitro mode...

Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.