Person: Rajaiya, Jaya
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Rajaiya
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Jaya
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Rajaiya, Jaya
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Publication Role of MyD88 in Adenovirus Keratitis(2016) Zhou, Xiaohong; Ramke, Mirja; Chintakuntlawar, Ashish V.; Lee, Jeong Yoon; Rajaiya, Jaya; Chodosh, JamesPattern recognition receptors (PRRs) are critical to the early detection and innate immune responses to pathogens. In particular, the TLR system and its associated adaptor proteins play essential roles in early host responses to infection. Epidemic keratoconjunctivitis, caused by the human adenovirus, is a severe ocular surface infection associated with corneal inflammation (stromal keratitis). We previously showed that adenovirus capsid was a key molecular pattern in adenovirus keratitis, with viral DNA playing a lesser role. We have now investigated the role of the adaptor molecule MyD88 in a mouse model of adenovirus keratitis in which there is no viral replication. In MyD88−/− mice infected with human adenovirus type 37, clinical keratitis was markedly reduced, along with infiltration of CD45+ cells, and expression of inflammatory cytokines. Reduction of inflammatory cytokines was also observed in infected primary human corneal fibroblasts pretreated with a MyD88 inhibitory peptide. Keratitis similar to wild type mice was observed in TLR2, TLR9, and IL-1R knockout mice, but was reduced in TLR2/9 double knockout mice, consistent with synergy of TLR2 and TLR9 in the response to adenovirus infection. MyD88 co-immunoprecipitated with Src kinase in mice corneas and in human corneal fibroblasts infected with adenovirus, and MyD88 inhibitory peptide reduced Src phosphorylation, linking MyD88 activation to inflammatory gene expression through a signaling cascade previously shown to be directed by Src. Our findings reveal a critical role for the PRRs TLR2 and 9, and their adaptor protein MyD88, in corneal inflammation upon adenovirus infection.Publication Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection(American Society for Microbiology, 2018) Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.; Rajaiya, Jaya; Chodosh, JamesABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE: Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.Publication Genomic analysis of a large set of currently—and historically—important human adenovirus pathogens(Nature Publishing Group UK, 2018) Ismail, Ashrafali M.; Cui, Tiange; Dommaraju, Kalpana; Singh, Gurdeep; Dehghan, Shoaleh; Seto, Jason; Shrivastava, Susmita; Fedorova, Nadia B.; Gupta, Neha; Stockwell, Timothy B.; Madupu, Ramana; Heim, Albert; Kajon, Adriana E.; Romanowski, Eric G.; Kowalski, Regis P.; Malathi, Jambulingam; Therese, Kuzhanthai L.; Madhavan, Hajib Narahari; Zhang, Qiwei; Ferreyra, Leonardo J.; Jones, Morris S.; Rajaiya, Jaya; Dyer, David W.; Chodosh, James; Seto, DonaldHuman 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.Publication Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells(Public Library of Science, 2013) Yousuf, Mohammad A.; Zhou, Xiaohong; Mukherjee, Santanu; Chintakuntlawar, Ashish V.; Lee, Jeong Yoon; Ramke, Mirja; Chodosh, James; Rajaiya, JayaThe 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.Publication The 5′UTR in human adenoviruses: leader diversity in late gene expression(Nature Publishing Group UK, 2017) Ramke, Mirja; Lee, Jeong Yoon; Dyer, David W.; Seto, Donald; Rajaiya, Jaya; Chodosh, JamesHuman adenoviruses (HAdVs) shut down host cellular cap-dependent mRNA translation while initiating the translation of viral late mRNAs in a cap-independent manner. HAdV 5′ untranslated regions (5′UTRs) are crucial for cap-independent initiation, and influence mRNA localization and stability. However, HAdV translational regulation remains relatively uncharacterized. The HAdV tripartite leader (TPL), composed of three introns (TPL 1–3), is critical to the translation of HAdV late mRNA. Herein, we annotated and analyzed 72 HAdV genotypes for the HAdV TPL and another previously described leader, the i-leader. Using HAdV species D, type 37 (HAdV-D37), we show by reverse transcription PCR and Sanger sequencing that mRNAs of the HAdV-D37 E3 transcription unit are spliced to the TPL. We also identified a polycistronic mRNA for RID-α and RID-β. Analysis of the i-leader revealed a potential open reading frame within the leader sequence and the termination of this potential protein in TPL3. A potential new leader embedded within the E3 region was also detected and tentatively named the j-leader. These results suggest an underappreciated complexity of post-transcriptional regulation, and the importance of HAdV 5′UTRs for precisely coordinated viral protein expression along the path from genotype to phenotype.Publication Epidemic Keratoconjunctivitis-Causing Adenoviruses Induce MUC16 Ectodomain Release To Infect Ocular Surface Epithelial Cells(American Society for Microbiology, 2016) Menon, Balaraj B.; Zhou, Xiaohong; Spurr-Michaud, Sandra; Rajaiya, Jaya; Chodosh, James; Gipson, IleneABSTRACT Human adenoviruses (HAdV), species D in particular (HAdV-D), are frequently associated with epidemic keratoconjunctivitis (EKC). Although the infection originates at the ocular surface epithelium, the mechanisms by which HAdV-Ds bypass the membrane-associated mucin (MAM)-rich glycocalyx of the ocular surface epithelium to trigger infection and inflammation remain unknown. Here, we report that an EKC-causing adenovirus (HAdV-D37), but not a non-EKC-causing one (HAdV-D19p), induces ectodomain release of MUC16—a MAM with barrier functions at the ocular surface—from cultured human corneal and conjunctival epithelial cells. HAdV-D37, but not HAdV-D19p, is also found to decrease the glycocalyx barrier function of corneal epithelial cells, as determined by rose bengal dye penetrance assays. Furthermore, results from quantitative PCR (qPCR) amplification of viral genomic DNA using primers specific to a conserved region of the E1B gene show that, in comparison to infection by HAdV-D19p, infection by HAdV-D37 is significantly increased in corneal epithelial cells. Collectively, these results point to a MUC16 ectodomain release-dependent mechanism utilized by the EKC-causing HAdV-D37 to initiate infection at the ocular surface. These findings are important in terms of understanding the pathogenesis of adenoviral keratoconjunctivitis. Similar MAM ectodomain release mechanisms may be prevalent across other mucosal epithelia in the body (e.g., the airway epithelium) that are prone to adenoviral infection. IMPORTANCE: Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause infections across all mucosal tissues in the body. At the ocular surface, HAdVs cause keratoconjunctivitis (E. Ford, K. E. Nelson, and D. Warren, Epidemiol Rev 9:244–261, 1987, and C. M. Robinson, D. Seto, M. S. Jones, D. W. Dyer, and J. Chodosh, Infect Genet Evol 11:1208–1217, 2011, doi:10.1016/j.meegid.2011.04.031)—a highly contagious infection that accounts for nearly 60% of conjunctivitis cases in the United States (R. P. Sambursky, N. Fram, and E. J. Cohen, Optometry 78:236–239, 2007, doi:10.1016/j.optm.2006.11.012, and A. M. Pihos, J Optom 6:69–74, 2013, doi:10.1016/j.optom.2012.08.003). The infection begins with HAdV entry within ocular surface epithelial cells; however, the mechanisms used by HAdVs to transit the otherwise protective mucosal barrier of ocular surface epithelial cells prior to entry remain unknown. Here, we report that the highly virulent keratoconjunctivitis-causing HAdV-D37 induces release of the extracellular domain (ectodomain) of MUC16, a major component of the mucosal barrier of ocular surface epithelial cells, prior to infecting underlying cells. Currently, there is no specific treatment for controlling this infection. Understanding the early steps involved in the pathogenesis of keratoconjunctivitis and using this information to intercept adenoviral entry within cells may guide the development of novel strategies for controlling the infection.Publication Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus(American Society of Microbiology, 2013) Robinson, Christopher; Zhou, Xiaohong; Rajaiya, Jaya; Yousuf, Mohammad Abu; Singh, Gurdeep; DeSerres, Joshua J.; Walsh, Michael P.; Wong, Sallene; Seto, Donald; Dyer, David W.; Chodosh, James; Jones, Morris S.For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses.Publication Molecular evolution of human adenoviruses(Nature Publishing Group, 2013) Robinson, Christopher; Singh, Gurdeep; Lee, Jeong Yoon; Dehghan, Shoaleh; Rajaiya, Jaya; Liu, Elizabeth B.; Yousuf, Mohammad Abu; Betensky, Rebecca; Jones, Morris S.; Dyer, David W.; Seto, Donald; Chodosh, JamesThe 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.Publication Specific NFκB Subunit Activation and Kinetics of Cytokine Induction in Adenoviral Keratitis(Molecular Vision, 2009) Rajaiya, Jaya; Sadeghi, Neda; Chodosh, JamesPurpose: Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea. Methods: Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. Results: Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFκB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFκB p65 homodimer or NFκB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFκB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection. Conclusion: The kinetics of NFκB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFκB subunits.Publication Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis(Public Library of Science, 2010) Chintakuntlawar, Ashish V.; Zhou, Xiaohong; Rajaiya, Jaya; Chodosh, JamesHuman 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.