Jaya Rajaiya

Molecular evolution of human adenoviruses

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.

Computational analysis of human adenovirus type 22 provides evidence for recombination among species D human adenoviruses in the penton base gene.

ABSTRACT Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.

Induction of Immunoglobulin Heavy-Chain Transcription through the Transcription Factor Bright Requires TFII-I

ABSTRACT 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, Brutons 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 Brights 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.

Analysis of Human Adenovirus Type 19 Associated with Epidemic Keratoconjunctivitis and its Reclassification as Adenovirus Type 64

PURPOSE Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen. METHODS High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry. RESULTS HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64. CONCLUSIONS 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.

Bruton's Tyrosine Kinase Regulates Immunoglobulin Promoter Activation in Association with the Transcription Factor Bright

ABSTRACT 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 Brutons 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.

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression

BackgroundHuman adenovirus type 19 (HAdV-19) is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of p38 mitogen-activated protein kinase (MAPK) in HAdV-19 infection, with particular attention to the role of p38 MAPK in the transcriptional control of interleukin-8 (IL-8), a chemokine previously shown to be central to the initiation of adenovirus keratitis.ResultsWe found that infection of corneal cells with HAdV-19 led to activation of p38 MAPK and its downstream targets, HSP-27 and ATF-2, within 15 to 30 minutes post-infection. Infection also induced phosphorylation of IκB and NFκB in a p38 MAPK-dependent fashion. Furthermore, HAdV-19 induced an interaction between p38 MAPK and NFκB-p65, followed by nuclear translocation of activated NFκB-p65 and its binding to the IL-8 promoter. The interaction between p38 MAPK and NFκB-p65 was inhibited in concentration-dependent fashion by SB203580, a chemical inhibitor of p38 MAPK, but not by SP600125, an inhibitor of JNK – another MAPK implicated in chemokine expression by HAdV-19 infected cells. IL-8 gene expression in HAdV-19 infection was significantly reduced in the presence of sequence-specific p38 MAPK siRNA but not control siRNA.ConclusionThese results provide the first direct evidence for transcriptional regulation of IL-8 in HAdV-19 infected cells through the activation of the p38 MAPK signaling pathway. The p38 MAPK pathway may play a biologically important role in regulation of IL-8 gene expression in the adenovirus-infected cornea.

Caveolin-1 associated adenovirus entry into human corneal cells.

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.

The E3 CR1-gamma gene in human adenoviruses associated with epidemic keratoconjunctivitis

Human adenovirus species D type 37 (HAdV-D37) is an important etiologic agent of epidemic keratoconjunctivitis. Annotation of the whole genome revealed an open reading frame (ORF) in the E3 transcription unit predicted to encode a 31.6kDa protein. This ORF, also known as CR1-γ, is predicted to be an integral membrane protein containing N-terminal signal sequence, luminal, transmembrane, and cytoplasmic domains. HAdV-D19 (C), another viral pathogen causing epidemic keratoconjunctivitis, contains an ORF 100% identical to its HAdV-D37 homologue but only 66% identical to other HAdV-D homologues. Kinetics of RNA expression and confirmation of splicing to the adenovirus tripartite leader sequence suggest a role for the protein product of CR1-γ in the late stages of the viral replication cycle. Confocal microscopy is consistent with expression in the cytoplasm. Sequence analysis reveals a hypervariable luminal domain and a conserved cytoplasmic domain. The luminal domain is predicted to contain multiple N-glycosylation sites. The cytoplasmic domain contains a putative protein kinase C phosphorylation site and potential YXXϕ and dileucine (LL) motifs suggesting a potential role in modification of host proteins.

Recombination of the epsilon determinant and corneal tropism: Human adenovirus species D types 15, 29, 56, and 69

Viruses within human adenovirus species D (HAdV-D) infect epithelia at essentially every mucosal site. Hypervariable loops 1 and 2 of the hexon capsid protein contain epitopes that together form the epsilon determinant for serum neutralization. We report our analyses comparing HAdV-D15, 29, 56, and the recently identified type 69, each with highly similar hexons and the same serum neutralization profile, but otherwise disparate genomes. Of these, only HAdV-D type 56 is associated with epidemic keratoconjunctivitis (EKC), a severe infection of ocular surface epithelium and underlying corneal stroma. In the mouse adenovirus keratitis model, all four viruses induced inflammation. However, HAdV-D56 entry into human corneal epithelial cells and fibroblasts in vitro dramatically exceeded that of the other three viruses. We conclude that the hexon epsilon determinant is not a prime contributor to corneal tropism.

Ultrastructure of adenovirus keratitis.

PURPOSE We determined the ultrastructure of mouse adenovirus keratitis, a model for human adenovirus keratitis. METHODS Adenovirus keratitis was induced in C57Bl/6j mice by intrastromal injection of human adenovirus species D type 37 (HAdV-D37) with a heat-pulled, glass, micropipette needle under compressed air. At select time points after infection, mice were euthanized and their corneas removed, fixed, and sectioned at 70-nm thickness for electron microscopy. RESULTS Injection of HAdV-D37 into the mouse corneal stroma placed virus predominantly in the pericellular corneal stromal matrix. Virus was seen bound to and entering stromal cells at 1 and 2 hours after infection, respectively. Cell membrane transit by virus was seen to involve two distinct structures resembling caveolae and macropinosomes. However, later during infection intracellular virus was not seen within membrane-bound organelles. By 8 hours after infection, intracellular virus had accumulated into densely packed, perinuclear arrays. Virus disassembly was not obvious at any time point after infection. Infiltrating neutrophils seen by one day after infection had engulfed degraded stromal cells by 4 days after infection. CONCLUSIONS By transmission electron microscopy, injected HAdV-D37 readily enters stromal cells in the C57Bl/6j mouse cornea and induces stromal inflammation, as was shown previously by light microscopy. However, electron microscopy also revealed dense, static arrays of intracytoplasmic virus, suggesting a block in viral capsid disassembly and viral DNA nuclear entry. These findings may explain why human adenoviruses do not replicate in the mouse corneal stroma.